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Sample records for agarose gel matrix

  1. Agarose gel electrophoresis.

    PubMed

    Smith, D R

    1993-01-01

    After digestion of DNA with a restriction enzyme (Chapter 50), it is usually necessary, for both preparative and analytical purposes, to separate and visualize the products. In most cases, where the products are between 200 and 20,000 bp long, this is achieved by agarose gel electrophoresis. Agarose is a linear polymer that is extracted from seaweed and sold as a white powder. The powder is melted in buffer and allowed to cool, whereby the agarose forms a gel by hydrogen bonding. The hardened matrix contains pores, the size of which depends on the concentration of agarose. The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.3 to 3%. Many different apparatus arrangements have been devised to run agarose gels; for example, they can be run horizontally or vertically, and the current can be conducted by wicks or the buffer solution. However, today, the "submarine" gel system is almost universally used. In this method, the agarose gel is formed on a supporting plate, and then the plate is submerged into a tank containing a suitable electrophoresis buffer. Wells are preformed in the agarose gel with the aid of a "comb" that is inserted into the cooling agarose before the agarose has gelled. Into these wells are loaded the sample to be analyzed, which has been mixed with a dense solution (a loading buffer) to ensure that the sample sinks into the wells.

  2. A new agarose matrix for single-strand conformation polymorphism (SSCP), heteroduplex (HTX), and gel shift analyses

    SciTech Connect

    Dumais, M.M.; White, H.W.; Rashid, M.R.

    1994-09-01

    Detection of mutation, by SSCP or heteroduplex analysis, is important in medical genetics and oncology. Analysis of DNA binding proteins is a powerful tool in molecular biology research. Traditionally, these methods are performed using nondenaturing gel electrophoresis on poly-acrylamide or polyacrylamide-type matrices. Here we report the development of a new agarose gel matrix that can be used for all three methods. SSCP analyses were performed using the prototype agarose gel matrix for wild-type, polymorphic, and mutant samples from c-Kras exon 12, p53 exons 8 and 9, and HOX2B. We performed SSCP analyses using both isotopic and nonisotopic methods. We also analyzed the samples by deliberate HTX formation and subsequent gel analysis. Using the prototype agarose matrix, we detected single and multiple DNA sequence variants in 150-350 bp fragments with an efficiency comparable to polyacrylamide gels run under similar conditions. For SSCP and HTX assays, we achieved optimal resolution in gels run in vertical formats. However, some HTX samples could be resolved in horizontal gel systems. In addition, based on our studies, we have developed a useful battery of controls and standards for quality control of SSCP and HTX assays. We analyzed several different DNA/protein complexes (SP1, AP2, and octamer binding protein) using the prototype agarose matrix. We obtained good resolution in both vertical and horizontal gel formats. The horizontal gel system is generally superior for this application, due to its ease of use and slightly better resolution. This new prototype gel matrix offers an alternative for researchers performing analyses that previously could only be done on polyacrylamide-type gel matrices. For some applications, this new matrix offers the ease of horizontal gel casting. For all applications, this matrix offers the safety of a nontoxic system and the reproducibility of a thermally gelling system.

  3. A new agarose gel model

    SciTech Connect

    Hasenfeld, A.; Pepke, E.; Lim, H.A.; Cantor, C.R.

    1993-12-31

    A new agarose gel model is introduced, which corresponds to what the authors believe agarose gels look like microscopically. While the scientific literature is filled with studies of the microscopic structure of agarose, the fact remains that there is no unambiguous and exact model of its underlying structure. Given this, the authors are left to construct their own model numerically.

  4. Mechanical and structural contribution of non-fibrillar matrix in uniaxial tension: a collagen-agarose co-gel model.

    PubMed

    Lake, Spencer P; Barocas, Victor H

    2011-07-01

    The mechanical role of non-fibrillar matrix and the nature of its interaction with the collagen network in soft tissues remain poorly understood, in part because of the lack of a simple experimental model system to quantify these interactions. This study's objective was to examine mechanical and structural properties of collagen-agarose co-gels, utilized as a simplified model system, to understand better the relationships between the collagen network and non-fibrillar matrix. We hypothesized that the presence of agarose would have a pronounced effect on microstructural reorganization and mechanical behavior. Samples fabricated from gel solutions containing 1.0 mg/mL collagen and 0, 0.125, or 0.25% w/v agarose were evaluated via scanning electron microscopy, incremental tensile stress-relaxation tests, and polarized light imaging. While the incorporation of agarose did not dramatically alter collagen network morphology, agarose led to concentration-dependent changes in mechanical and structural properties. Specifically, resistance of co-gels to volume change corresponded with differences in fiber reorientation and elastic/viscoelastic mechanics. Results demonstrate strong relationships between tissue properties and offer insight into behavior of tissues of varying Poisson's ratio and fiber kinematics. Results also suggest that non-fibrillar material may have significant effects on properties of artificial and native tissues even in tension, which is generally assumed to be collagen dominated.

  5. Recycling of superfine resolution agarose gel.

    PubMed

    Seng, T-Y; Singh, R; Faridah, Q Z; Tan, S-G; Alwee, S S R S

    2013-07-08

    Genetic markers are now routinely used in a wide range of applications, from forensic DNA analysis to marker-assisted plant and animal breeding. The usual practice in such work is to extract the DNA, prime the markers of interest, and sift them out by electrically driving them through an appropriate matrix, usually a gel. The gels, made from polyacrylamide or agarose, are of high cost, limiting their greater applications in molecular marker work, especially in developing countries where such technology has great potential. Trials using superfine resolution (SFR) agarose for SSR marker screening showed that it is capable of resolving SSR loci and can be reused up to 14 times, thus greatly reducing the cost of each gel run. Furthermore, for certain applications, low concentrations of agarose sufficed and switching to lithium borate buffer, instead of the conventional Tris-borate-ethylenediaminetetraacetic acid buffer, will further save time and cost. The 2.5% gel was prepared following the Agarose SFR(TM) manual by adding 2.5 g agarose powder into 100 mL 1X lithium borate buffer in a 250-mL flask with rapid stirring. Two midigels (105 x 83 mm, 17 wells) or 4 minigels (50 x 83 mm, 8 wells), 4 mm thickness can be prepared from 100 mL gel solution. A total of 1680 PCR products amplified using 140 SSR markers from oil palm DNA samples were tested in this study using SFR recycled gel. As average, the gel can be recycled 8 times with good resolution, but can be recycled up to 14 times before the resolutions get blurred.

  6. Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels.

    PubMed

    Nims, Robert J; Cigan, Alexander D; Albro, Michael B; Hung, Clark T; Ateshian, Gerard A

    2014-06-27

    Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-β3 supplementation. TGF-β3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-β3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

  7. Agarose gel electrophoresis for the separation of DNA fragments.

    PubMed

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  8. Nondenaturing agarose gel electrophoresis of RNA.

    PubMed

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W

    2010-06-01

    INTRODUCTION Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. There are two common types of gel: polyacrylamide and agarose. For most applications involving RNAs of < or =600 nucleotides, denaturing acrylamide gels are most appropriate. In contrast, agarose gels are generally used to analyze RNAs of > or =600 nucleotides, and are especially useful for analysis of mRNAs (e.g., by Northern blotting). RNA analysis on agarose gels is essentially identical to DNA analysis (except that the gel boxes used must be dedicated to RNA work or to other ribonuclease-free work). Here we describe the use of straightforward Tris borate, EDTA (TBE) gels for routine analysis. These gels are appropriate for determining the quantity and integrity of RNA before using it for other applications. This procedure should not be used to determine size with accuracy, because the RNA will not remain in its extended state throughout the run.

  9. On-bead expression of recombinant proteins in an agarose gel matrix coated on a glass slide.

    PubMed

    Lee, Kyung-Ho; Lee, Ka-Young; Byun, Ju-Young; Kim, Byung-Gee; Kim, Dong-Myung

    2012-05-07

    A system for expression and in situ display of recombinant proteins on a microbead surface is described. Biotinylated PCR products were immobilized on microbead surfaces, which were then embedded in a gel matrix and supplied with translation machinery and substrates. Upon the incubation of the gel matrix, target proteins encoded on the bead-immobilized DNA were expressed and captured on the same bead, thus allowing bead-mediated linkage of DNA and encoded proteins. The new method combines the simplicity and convenience of solid-phase separation of genetic information with the benefits of cell-free protein synthesis, such as instant translation of genetic information, unrestricted substrate accessibility and flexible assay configuration design.

  10. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.

    PubMed

    Stellwagen, Nancy C

    2009-06-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

  11. Bleach gel: a simple agarose gel for analyzing RNA quality.

    PubMed

    Aranda, Patrick S; LaJoie, Dollie M; Jorcyk, Cheryl L

    2012-01-01

    RNA-based applications requiring high-quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the 'bleach gel' is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.

  12. Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality

    PubMed Central

    Aranda, Patrick S.; LaJoie, Dollie M.; Jorcyk, Cheryl L.

    2013-01-01

    RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality. PMID:22222980

  13. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Guarrotxena, Nekane; Braun, Gary

    2012-10-01

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  14. Blinking suppression of single quantum dots in agarose gel

    SciTech Connect

    Ko, H. C.; Yuan, C. T.; Tang, Jau; Lin, S. H.

    2010-01-04

    Fluorescence blinking is commonly observed in single molecule/particle spectroscopy, but it is an undesirable feature in many applications. We demonstrated that single CdSe/ZnS quantum dots in agarose gel exhibited suppressed blinking behavior. In addition, the long-time exponential bending tail of the power-law blinking statistics was found to be influenced by agarose gel concentration. We suggest that electron transfer from the light state to the dark state might be blocked due to electrostatic surrounding of gel with inherent negatively charged fibers.

  15. Separation of long RNA by agarose-formaldehyde gel electrophoresis.

    PubMed

    Mansour, Farrah H; Pestov, Dimitri G

    2013-10-01

    We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative "pK-matched" buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.

  16. Electric birefrigence imaging of DNA in agarose electrophoresis gels

    SciTech Connect

    Lanan, M.

    1992-01-01

    Electric birefringence imaging (EBI) provides sensitive, non-invasive detection of double-stranded DNA in agarose gels. Quasi-monochromatic, visible light is transmitted through an electrophoresis gel which is placed between plastic film polarizers. A slow-scan video camera equipped with a 12 bit A/D converter records the images. Under electrophoresis running conditions, hydrodynamically-induced gel distortion is shown to be the major source of birefringence for fragments smaller than 23 kbp. The birefringence generated approximates the DNA concentration gradient in the electric field direction. The stress-optic coefficient of 1% agarose gel is measured by mechanical compression and used to evaluate the magnitude of the induced stress on the gel during electrophoresis. Multi-linear regression analysis is used to quantitatively test the model for EBI signals. Birefringence attributed to localized electrokinetic gel distortion and to intrinsic DNA birefringence is studied by fitting ethidium bromide fluorescence profiles to EBI results. Fluorescence polarization imaging is used to assess the influence of localized gel distortion on nucleic acid orientation across a fragment band. It is shown that DNA aligns parallel, on average, with an applied electric field independent of its location within a band. Both EBI sensitivity and quantitation are improved through image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions. Under standard electrophoresis conditions, detection limits of 8 ng DNA per well are obtained in hydroxyethylated agarose without signal averaging. Maintaining constant gel temperature is shown to improve the quality of the images. Stress patterns in agarose gels during DC and field-inversion gel electrophoresis (FIGE) of nucleic acid fragments of varying sizes are mapped using EBI. In addition, online EBI monitoring during FIGE of megabase pair DNA size standards is demonstrated.

  17. Enhanced detection of gold nanoparticles in agarose gel electrophoresis.

    PubMed

    Hasenoehrl, Carina; Alexander, Colleen M; Azzarelli, Nicholas N; Dabrowiak, James C

    2012-04-01

    Gel electrophoresis is a powerful tool in gold nanoparticle (AuNP) research. While the technique is sensitive to the size, charge, and shape of particles, its optimal performance requires a relatively large amount of AuNP in the loading wells for visible detection of bands. We here describe a novel and more sensitive method for detecting AuNPs in agarose gels that involves staining the gel with the common organic fluorophore fluorescein, to produce AuNP band intensities that are linear with nanoparticle concentration and almost an order of magnitude larger than those obtained without staining the gel.

  18. Nondenaturing electrophoresis of lipoproteins in agarose and polyacrylamide gradient gels

    SciTech Connect

    Shore, V.G.

    1989-12-19

    The plasma lipoproteins frequently are classified according to density and/or electrophoretic mobility. The lipoprotein classes differ characteristically also in particle size and apolipoprotein composition. Each class is heterogeneous in size and composition as well. Nondenaturing electrophoresis in agarose gels and polyacrylamide gradient gels are complementary analytical methods for classification of lipoproteins and determining distribution profiles of the major classes. In addition, gradient gel electrophoresis (GGE) has a high resolving capability for subfractionating each class according to particle size. Combination of gel electrophoresis with immunoblotting yields information on heterogeneity in apolipoprotein distribution. 14 refs., 6 figs., 3 tabs.

  19. Function, structure, and stability of enzymes confined in agarose gels.

    PubMed

    Kunkel, Jeffrey; Asuri, Prashanth

    2014-01-01

    Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.

  20. Anomalous diffusion of poly(ethylene oxide) in agarose gels.

    PubMed

    Brenner, Tom; Matsukawa, Shingo

    2016-11-01

    We report on the effect of probe size and diffusion time of poly(ethylene) oxide in agarose gels. Time-dependence of the diffusion coefficient, reflecting anomalous diffusion, was observed for poly(ethylene) oxide chains with hydrodynamic radii exceeding about 20nm at an agarose concentration of 2%. The main conclusion is that the pore distribution includes pores that are only several nm across, in agreement with scattering reports in the literature. Interpretation of the diffusion coefficient dependence on the probe size based on a model of entangled rigid rods yielded a rod length of 72nm.

  1. Properties of cellulase immobilized on agarose gel with spacer

    SciTech Connect

    Chim-anage, P.; Kashiwagi, Y.; Magae, Y.; Ohta, T.; Sasaki, T.

    1986-12-01

    Cellulase produced by fungus Trichoderma viride was immobilized on agarose beads (Sepharose 4B) activated by cyanogen bromide and also on activated agarose beads that contained spacer arm (activated Ch-Sepharose 4B and Affi-Gel 15). The CMCase activity retained by immobilized cellulase on activated Sepharose containing the spacer tended to be higher than that immobilized without spacer, although the extent of protein immobilization was lower. Also, the higher substrate specificity for cellulase immobilized on beads with spacer was obtained for cellobiose, acid-swollen cellulose, or cellulose powder. The hydrolysis product from their substrates was mainly glucose. 10 references.

  2. Posing for a picture: vesicle immobilization in agarose gel

    PubMed Central

    Lira, Rafael B.; Steinkühler, Jan; Knorr, Roland L.; Dimova, Rumiana; Riske, Karin A.

    2016-01-01

    Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs. PMID:27140695

  3. Posing for a picture: vesicle immobilization in agarose gel

    NASA Astrophysics Data System (ADS)

    Lira, Rafael B.; Steinkühler, Jan; Knorr, Roland L.; Dimova, Rumiana; Riske, Karin A.

    2016-05-01

    Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.

  4. Two-dimensional agarose gel electrophoresis of DNA topoisomers.

    PubMed

    Roca, Joaquim

    2009-01-01

    The electrophoretic velocity of a duplex DNA ring is mainly determined by its overall shape. Consequently, DNA topoisomers of opposite supercoiling handedness can have identical gel velocity, and topoisomers highly supercoiled cannot be separated beyond some point. These problems are overcome by two-dimensional agarose gel electrophoresis, which involves two successive electrophoresis steps in one gel slab. The first and second electrophoresis steps are conducted in orthogonal directions with different concentrations of DNA intercalating agents. These compounds alter the overall shape of the DNA and, thereby, change the relative mobility of individual DNA topoisomers.

  5. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  6. Pore size of agarose gels by atomic force microscopy.

    PubMed

    Pernodet, N; Maaloum, M; Tinland, B

    1997-01-01

    The pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the "unperturbed" gel without invasive treatment. The pore size a as a function of gel concentration C shows a power law dependence a approximately C-gamma, where gamma lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration increases.

  7. A polarized photobleaching study of DNA reorientation in agarose gels

    SciTech Connect

    Scalettar, B.A.; Klein, M.P. ); Selvin, P.R.; Hearst, J.E. Univ. of California, Berkeley ); Axelrod, D. )

    1990-05-22

    Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that reversible photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.

  8. Disease proteomics of high-molecular-mass proteins by two-dimensional gel electrophoresis with agarose gels in the first dimension (Agarose 2-DE).

    PubMed

    Oh-Ishi, Masamichi; Maeda, Tadakazu

    2007-04-15

    Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW>100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification.

  9. Modified gel preparation for distinct DNA fragment analysis in agarose gel electrophoresis.

    PubMed

    Lee, S V; Bahaman, A R

    2010-08-01

    Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.

  10. [Preparation, characterization and surface-enhanced Raman properties of agarose gel/gold nanoparticles hybrid].

    PubMed

    Ma, Xiao-yuan; Liu, Ying; Wang, Zhou-ping

    2014-08-01

    Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Naniocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis-NIR absorption spectroscopy. Experimental data indicated a uniform distribution of gold nanoparticles adsorbed on agarose gel network And the excellent optical absorption properties were shown. Based on the swelling-contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nano-composites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules Nile blue A. Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal.

  11. Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability

    PubMed Central

    Suzawa, Yoshika; Kubo, Norihiko; Iwai, Soichi; Yura, Yoshiaki; Ohgushi, Hajime; Akashi, Mitsuru

    2015-01-01

    Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects. PMID:26110392

  12. Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability.

    PubMed

    Suzawa, Yoshika; Kubo, Norihiko; Iwai, Soichi; Yura, Yoshiaki; Ohgushi, Hajime; Akashi, Mitsuru

    2015-06-23

    Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

  13. Response surface methodology-based optimisation of agarose gel electrophoresis for screening and electropherotyping of rotavirus.

    PubMed

    Mishra, Vikas; Nag, Vijaya Lakshmi; Tandon, Ritu; Awasthi, Shally

    2010-04-01

    Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l(-1) ionic strength of Tris base-boric acid-ethylenediamine tetraacetic acid buffer (1.4x) and 4.31 h of electrophoresis at 4.6 V cm(-1) electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice.

  14. Agarose gel structure using atomic force microscopy: gel concentration and ionic strength effects.

    PubMed

    Maaloum, M; Pernodet, N; Tinland, B

    1998-07-01

    Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.

  15. Preparation of gold nanoparticles-agarose gel composite and its application in SERS detection

    NASA Astrophysics Data System (ADS)

    Ma, Xiaoyuan; Xia, Yu; Ni, Lili; Song, Liangjing; Wang, Zhouping

    2014-03-01

    Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis-NIR absorption spectroscopy. Based on the swelling-contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection.

  16. Preparation of gold nanoparticles-agarose gel composite and its application in SERS detection.

    PubMed

    Ma, Xiaoyuan; Xia, Yu; Ni, Lili; Song, Liangjing; Wang, Zhouping

    2014-01-01

    Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis-NIR absorption spectroscopy. Based on the swelling-contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection.

  17. An agarose-gel based method for transporting cell lines.

    PubMed

    Yang, Lingzhi; Li, Chufang; Chen, Ling; Li, Zhiyuan

    2009-12-16

    Cryopreserved cells stored in dry ice or liquid nitrogen is the classical method for transporting cells between research laboratories in different cities around the world in order to maintain cell viability. An alternative method is to ship the live cells in flasks filled with cell culture medium. Both methods have limitations of either a requirement on special shipping container or short times for the cells to survive on the shipping process. We have recently developed an agarose gel based method for directly transporting the live adherent cells in cell culture plates or dishes in ambient temperature. This convenient method simplifies the transportation of live cells in long distance that can maintain cells in good viability for several days.

  18. Cloning of DNA fragments: ligation reactions in agarose gel.

    PubMed

    Furtado, Agnelo

    2014-01-01

    Ligation reactions to ligate a desired DNA fragment into a vector can be challenging to beginners and especially if the amount of the insert is limiting. Although additives known as crowding agents, such as PEG 8000, added to the ligation mixes can increase the success one has with ligation reactions, in practice the amount of insert used in the ligation can determine the success or the failure of the ligation reaction. The method described here, which uses insert DNA in gel slice added directly into the ligation reaction, has two benefits: (a) using agarose as the crowding agent and (b) reducing steps of insert purification. The use of rapid ligation buffer and incubation of the ligation reaction at room temperature greatly increase the efficiency of the ligation reaction even for blunt-ended ligation.

  19. Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

    PubMed

    Arimura, Shin-ichiro; Kawahara, Ko-ichi; Biswas, Kamal Krishna; Abeyama, Kazuhiro; Tabata, Masashi; Shimoda, Toru; Ogomi, Daisuke; Matsusaki, Michiya; Kato, Shinya; Ito, Takashi; Sugihara, Kazumasa; Akashi, Mitsuru; Hashiguchi, Teruto; Maruyama, Ikuro

    2007-05-01

    We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

  20. Regenerative behavior of biomineral/agarose composite gels as bone grafting materials in rat cranial defects.

    PubMed

    Suzawa, Yoshika; Funaki, Takafumi; Watanabe, Junji; Iwai, Soichi; Yura, Yoshiaki; Nakano, Takayoshi; Umakoshi, Yukichi; Akashi, Mitsuru

    2010-06-01

    The main objective of this study was to evaluate the biological behavior of Hydroxyapatite (HAp)/agarose and calcium carbonate (CaCO3)/agarose composite gels by an alternate soaking process used for the treatment of surgically produced bone defects in rat cranium. We designed the following four groups: (i) HAp (HAp/agarose composite gel), (ii) CaCO3 (CaCO3/agarose composite gel), (iii) Agarose (bare agarose gel), and (iv) Defect (no filling materials). We subdivided (i) (ii) (iii) into two application types as a (I) Homogenized Group (homogenized materials) and a (II) Disk Group (disk shaped materials). We assessed samples by radiological and histological analyses 0, 4, and 8 weeks after implantation. The results indicated that the composite gels showed higher radiopacity in microfocus-computed tomography (muCT) images and showed higher volume in quantitative analyses using Dual Energy X-ray Absorptiometry (DEXA) and Peripheral Quantitative Computed Tomography (pQCT) than the Agarose and Defect groups. The histological examination showed characteristic images due to each application form. Consequently, HAp and CaCO3/agarose composite gels can be expected to accelerate the speed of producing more new bone associated with osteogenesis. These novel biomaterials play an important role as an alternative biocompatible and biodegradable bone grafting filler material for autogenous bone.

  1. Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers.

    PubMed

    Sitaras, Chris; Naghavi, Mahsa; Herrington, Muriel B

    2011-01-15

    Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)-agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS-agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS-agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS-agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.

  2. Effect of ultrasound on the separation of DNA fragments in agarose gel electrophoresis

    SciTech Connect

    Ma, Yinfa; Yeung, E.S. )

    1990-06-01

    Since its first use in 1966 interest in and the applications of electrophoresis of DNA fragments in agarose gel have grown rapidly. Nowadays, agarose gel electrophoresis has become a standard technique with high resolving power for the analysis of DNA structure, for example for the determination of the length of DNA fragments obtained by the action of restriction enzymes. The electrophoretic mobility ({mu}) of DNA fragments is influenced by various parameters-molecular weight, gel concentration, temperature, electric field, and DNA-agarose affinity. A comprehensive study of the influence of these main parameters has been reported. In this paper, the authors investigate a new effect on the electrophoretic mobility of DNA fragments in agarose gels, viz. the influence of ultrasound.

  3. Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis.

    PubMed

    Roncada, Paola; Cretich, Marina; Fortin, Riccardo; Agosti, Susanna; De Franceschi, Lucia; Greppi, Gian Franco; Turrini, Francesco; Carta, Franco; Turri, Stefano; Levi, Marinella; Chiari, Marcella

    2005-06-01

    A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.

  4. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    PubMed

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.

  5. Rapid extraction and structural characterization of biomolecules in agarose gels by laser desorption Fourier transform mass spectrometry

    SciTech Connect

    Dunphy, J.C.; Busch, K.L. ); Hettich, R.L.; Buchanan, M.V. )

    1993-05-15

    A method originally developed for the extraction of biomolecules from agarose gel slices has been utilized as a rapid means of isolating biological compounds from gels for subsequent structural characterization by matrix-assisted laser desorption-ionization Fourier transform mass spectrometry (MALDI/FTMS). This [open quotes]freeze-squeeze[close quotes] extraction method involves pressure extrusion of fluid from frozen gel slices and provides near 50% recovery of analyte in less than 5 min. Experiments were directed at examining the recovery efficiency of the extraction method using [sup 14]C-labeled adenosine monophosphate and investigating the effect of high buffer concentrations on the laser desorption mass spectra. When coupled with this extraction technique, MALDI/FTMS can be used to detect and identify biomolecules at the low picomole level in agarose gel slices. The accurate mass measurements and MS/MS capabilities of the FTMS were exploited to provide detailed structural information at the isomeric level for oligonucleotides electrophoresed into agarose gels. 41 refs., 5 figs., 1 tab.

  6. Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels.

    PubMed

    Cole, Kenneth D; Gaigalas, Adolfas; Akerman, Björn

    2006-11-01

    The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

  7. A composite agarose-polyacrylamide matrix as two-dimensional hard support for solid-phase protein assays.

    PubMed

    Krajewski, Wladyslaw A

    2016-03-15

    The solid-phase protein assays using blotting membranes as hard support do not allow achieving the low background and sensitivity of protein staining in clear gels. The membrane opacity complicates imaging of results on standard lab documentation systems. We describe a low-cost transparent matrix that can be used as an alternative to polymeric membranes for solid-phase assays. Protein samples are spotted onto a dry film of composite agarose-polyacrylamide matrix covering standard glass microscopic slides. After rehydration in protein-fixing solution, matrix with protein samples can be detached from glass support and stained as conventional protein polyacrylamide gels.

  8. Interpenetrating network formation in gellan--agarose gel composites.

    PubMed

    Amici, E; Clark, A H; Normand, V; Johnson, N B

    2000-01-01

    Thermal, mechanical, turbidity, and microscope evidence is provided which strongly suggests molecular interpenetrating network (IPN) formation by mixtures of the bacterial and seaweed polysaccharides gellan and agarose. There is no evidence for synergistic coupling of the networks, and simple phase separation (demixing) can definitely be ruled out. Some changes in the gellan gelling behavior are suggested, however, by the increased gellan effective concentrations implicit in cure curve data. The dependence of this effect on the agarose nominal concentration seems consistent with a previous model that focused on gelling parameters, and changes in these rather than real concentration effects. In large deformation mechanical tests, the influence of agarose added to gellan is to re-enforce the network (higher compression and shear moduli, higher stresses-to-break) without significantly changing the strain to break, or the gellan brittle failure mechanism.

  9. Impact of saccharides on the drying kinetics of agarose gels measured by in-situ interferometry

    NASA Astrophysics Data System (ADS)

    Mao, Bosi; Divoux, Thibaut; Snabre, Patrick

    2017-01-01

    Agarose gels are viscoelastic soft solids that display a porous microstructure filled with water at 90% w/w or more. Despite an extensive use in food industry and microbiology, little is known about the drying kinetics of such squishy solids, which suffers from a lack of time-resolved local measurements. Moreover, only scattered empirical observations are available on the role of the gel composition on the drying kinetics. Here we study by in-situ interferometry the drying of agarose gels of various compositions cast in Petri dishes. The gel thinning is associated with the displacement of interference fringes that are analyzed using an efficient spatiotemporal filtering method, which allows us to assess local thinning rates as low as 10 nm/s with high accuracy. The gel thinning rate measured at the center of the dish appears as a robust observable to quantify the role of additives on the gel drying kinetics and compare the drying speed of agarose gels loaded with various non-gelling saccharides of increasing molecular weights. Our work shows that saccharides systematically decrease the agarose gel thinning rate up to a factor two, and exemplifies interferometry as a powerful tool to quantify the impact of additives on the drying kinetics of polymer gels.

  10. Impact of saccharides on the drying kinetics of agarose gels measured by in-situ interferometry

    PubMed Central

    Mao, Bosi; Divoux, Thibaut; Snabre, Patrick

    2017-01-01

    Agarose gels are viscoelastic soft solids that display a porous microstructure filled with water at 90% w/w or more. Despite an extensive use in food industry and microbiology, little is known about the drying kinetics of such squishy solids, which suffers from a lack of time-resolved local measurements. Moreover, only scattered empirical observations are available on the role of the gel composition on the drying kinetics. Here we study by in-situ interferometry the drying of agarose gels of various compositions cast in Petri dishes. The gel thinning is associated with the displacement of interference fringes that are analyzed using an efficient spatiotemporal filtering method, which allows us to assess local thinning rates as low as 10 nm/s with high accuracy. The gel thinning rate measured at the center of the dish appears as a robust observable to quantify the role of additives on the gel drying kinetics and compare the drying speed of agarose gels loaded with various non-gelling saccharides of increasing molecular weights. Our work shows that saccharides systematically decrease the agarose gel thinning rate up to a factor two, and exemplifies interferometry as a powerful tool to quantify the impact of additives on the drying kinetics of polymer gels. PMID:28112236

  11. Two methods that facilitate autoradiography of small /sup 32/P-labeled DNA fragments following electrophoresis in agarose gels

    SciTech Connect

    Cockerill, P.N.

    1988-02-01

    Two methods which permit detection by autoradiography of small /sup 32/P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes.

  12. Agarose gel electrophoresis and polyacrylamide gel electrophoresis for visualization of simple sequence repeats.

    PubMed

    Anderson, James; Wright, Drew; Meksem, Khalid

    2013-01-01

    In the modern age of genetic research there is a constant search for ways to improve the efficiency of plant selection. The most recent technology that can result in a highly efficient means of selection and still be done at a low cost is through plant selection directed by simple sequence repeats (SSRs or microsatellites). The molecular markers are used to select for certain desirable plant traits without relying on ambiguous phenotypic data. The best way to detect these is the use of gel electrophoresis. Gel electrophoresis is a common technique in laboratory settings which is used to separate deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by size. Loading DNA and RNA onto gels allows for visualization of the size of fragments through the separation of DNA and RNA fragments. This is achieved through the use of the charge in the particles. As the fragments separate, they form into distinct bands at set sizes. We describe the ability to visualize SSRs on slab gels of agarose and polyacrylamide gel electrophoresis.

  13. Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability.

    PubMed Central

    White, Jeffrey A; Deen, William M

    2002-01-01

    Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices. PMID:11916864

  14. Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.

    PubMed

    Karim, Asima; Hall, Andrew C

    2017-05-01

    Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of

  15. Solvent-impregnated agarose gel liquid phase microextraction of polycyclic aromatic hydrocarbons in water.

    PubMed

    Loh, Saw Hong; Sanagi, Mohd Marsin; Wan Ibrahim, Wan Aini; Hasan, Mohamed Noor

    2013-08-09

    A new microextraction procedure termed agarose gel liquid phase microextraction (AG-LPME) combined with gas chromatography-mass spectrometry (GC-MS) was developed for the determination of selected polycyclic aromatic hydrocarbons (PAHs) in water. The technique utilized an agarose gel disc impregnated with the acceptor phase (1-octanol). The extraction procedure was performed by allowing the solvent-impregnated agarose gel disc to tumble freely in the stirred sample solution. After extraction, the agarose gel disc was removed and subjected to centrifugation to disrupt its framework and to release the impregnated solvent, which was subsequently withdrawn and injected into the GC-MS for analysis. Under optimized extraction conditions, the new method offered high enrichment factors (89-177), trace level LODs (9-14ngL(-1)) and efficient extraction with good relative recoveries in the range of 93.3-108.2% for spiked drinking water samples. AG-LPME did not exhibit any problems related to solvent dissolution, and it provided high extraction efficiencies that were comparable to those of hollow fiber liquid phase microextraction (HF-LPME) and significantly higher than those of agarose film liquid phase microextraction (AF-LPME). This technique employed a microextraction format and utilized an environmentally compatible solvent holder that supported the green chemistry concept.

  16. A simple, efficient, and economical method for recovering DNA from agarose gel.

    PubMed

    Fan, Chang-Fa; Mei, Xing-Guo

    2005-01-01

    A simple method of recovering DNA from agarose gel that is fast, inexpensive, and friendly both to operators and environment is described. Two rows of wells are made in an agarose gel, and a DNA sample is loaded into the well nearest to the negative pole for separation by electrophoresis. Recovery is accomplished by pipetting the DNA-containing TAE buffer from the well near the positive pole after target DNA fragments have migrated into the well. A recovery rate of up to 94 +/- 2.3% was observed with this method.

  17. Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium.

    PubMed

    Ishido, Tomomi; Ishikawa, Mitsuru; Hirano, Ken

    2010-05-01

    We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.

  18. Molecular stretching of long DNA in agarose gel using alternating current electric fields.

    PubMed Central

    Kaji, Noritada; Ueda, Masanori; Baba, Yoshinobu

    2002-01-01

    We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields. The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy. The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated. Stretching was only observed in the AC field when a frequency of approximately 10 Hz was used. The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used. Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3%. Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose. Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb). These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell. PMID:11751320

  19. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide.

    PubMed

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-12-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  20. Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Li, Jialiang; Yang, Yushi; Mao, Zhou; Huang, Wenjie; Qiu, Tong; Wu, Qingzhi

    2016-09-01

    In this work, a novel agarose gel electrophoresis strategy has been developed for separation of DNA fragments by doping graphene oxide (GO) into agarose gel. The results show that the addition of GO into agarose gel significantly improved the separation resolution of DNA fragments by increasing the shift distances of both the single DNA fragments and the adjacent DNA fragments and completely eliminating the background noise derived from the diffusion of the excessive ethidium bromide (EB) dye in the gel after electrophoresis. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO. These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules.

  1. Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

    PubMed

    Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

    2016-05-01

    Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE).

  2. Rapid recovery of DNA from agarose gel slices by coupling electroelution with monolithic SPE.

    PubMed

    Yu, Shengbing; Yang, Shuixian; Zhou, Ping; Zhou, Ke; Wang, Jing; Chen, Xiangdong

    2009-06-01

    An amino silica monolithic column prepared by in situ polymerization of tetraethoxysilane and N-(beta-aminoethyl)-gamma-aminopropyltriethoxysilane was firstly applied to recover DNA from agarose gel slices by coupling electroelution with monolithic SPE. DNA was electroeluted from the agarose gel slices onto the amino silica monolithic column. The DNA adsorbed on this monolithic column was then recovered using sodium phosphate solution at pH 10. The whole recovery procedure could be completed within 10 min because the use of amino silica monolithic column accelerated the DNA capture and facilitated the DNA release. Electroelution conditions, such as buffer pH, buffer concentration and applied voltage, were online optimized. The average yield for herring sperm DNA, pBR 322 DNA and lambda DNA recovered from 1.0% w/v agarose gel slices were 55+/-4, 50+/-6 and 42+/-7% (n=3), respectively. The polymerase chain reaction performance of pGM plasmid recovered from agarose gel slices demonstrated that the method could provide high-quality DNA for downstream processes. The combination of electroelution with monolithic SPE allows a rapid, simple and efficient DNA recovery method. This technique is especially useful for applications that need to purify small starting amounts of DNA.

  3. Local and average diffusion of nanosolutes in agarose gel: the effect of the gel/solution interface structure.

    PubMed

    Labille, Jérôme; Fatin-Rouge, Nicolas; Buffle, Jacques

    2007-02-13

    Fluorescence correlation spectroscopy (FCS) has been used to study the diffusion of nanometric solutes in agarose gel, at microscopic and macroscopic scales. Agarose gel was prepared and put in contact with aqueous solution. Several factors were studied: (i) the role of gel relaxation after its preparation, (ii) the specific structure of the interfacial zone and its role on the local diffusion coefficient of solutes, and (iii) the comparison between the local diffusion coefficient and the average diffusion coefficient in the gel. Fluorescent dyes and labeled biomolecules were used to cover a size range of solutes of 1.5 to 15 nm. Their transport through the interface from the solution toward the gel was modeled by the first Fick's law based on either average diffusion coefficients or the knowledge of local diffusion coefficients in the system. Experimental results have shown that, at the liquid/gel interface, a gel layer with a thickness of 120 microm is formed with characteristics significantly different from the bulk gel. In particular, in this layer, the porosity of agarose fiber network is significantly lower than in the bulk gel. The diffusion coefficient of solutes in this layer is consequently decreased for steric reasons. Modeling of solute transport shows that, in the bulk gel, macroscopic diffusion satisfactorily follows the classical Fick's diffusion laws. For the tested solutes, the local diffusion coefficients in the bulk gel, measured at microscopic scale by FCS, were equal, within experimental errors, to the average diffusion coefficients applicable at macroscopic scales (>or=mm). This confirms that anomalous diffusion applies only to solutes with sizes close to the gel pore size and at short time (

  4. Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations.

    PubMed Central

    Pluen, A; Netti, P A; Jain, R K; Berk, D A

    1999-01-01

    The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers. PMID:10388779

  5. Laser interferometric analysis of glucose and sucrose diffusion in agarose gel.

    PubMed

    Wąsik, Sławomir; Arabski, Michał; Dworecki, Kazimierz; Janoska, Joanna; Semaniak, Jacek; Szary, Karol; Slęzak, Andrzej

    2014-01-01

    The paper presents the investigation results of glucose and sucrose diffusion in agarose gel studied with laser interferometry method and the results of fluorescence analysis of the macroscopic gel structure. The diffusion kinetics of these substances released from aqueous solutions of a molar concentration of 0.05 M into the agarose solutions of concentrations of 0.5% and 3% in two gravitational configurations of measuring system was analysed. In the first configuration the solute diffused according, whereas in the second one - opposite to the gravitational force. The diffusion was analysed in the time period between 120 and 2400 s with a time interval of Δt = 120 s. We observed that the convective instabilities were damped well by the agarose gel, which gives the possibility of the interferometric studies of the diffusive transport for other substances in different gravitational configurations of the system. The time characteristics of glucose and sucrose fluxes in both configurations of the system and the gravitational polarisation coefficient values were obtained. The substantial differences in fluxes of glucose and sucrose diffused according and opposite to the gravitational force were observed. Additionally, we observed the differences between the diffusive fluxes of these substances in both configurations in dependence on the gel solution concentration (which is associated with gel porosity dependent on its concentration) and the kind of diffused substance.

  6. Plasmid DNA topology assayed by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, Jorge B; Martínez-Robles, María-Luisa; Hernández, Pablo; Krimer, Dora B

    2013-01-01

    Two-dimensional (2D) agarose gel electrophoresis is nowadays one of the best methods available to analyze DNA molecules with different masses and shapes. The possibility to use nicking enzymes and intercalating agents to change the twist of DNA during only one or in both runs, improves the capacity of 2D gels to discern molecules that apparently may look alike. Here we present protocols where 2D gels are used to understand the structure of DNA molecules and its dynamics in living cells. This knowledge is essential to comprehend how DNA topology affects and is affected by all the essential functions that DNA is involved in: replication, transcription, repair and recombination.

  7. DNA electrophoresis in agarose gels: A new mobility vs. DNA length dependence

    NASA Astrophysics Data System (ADS)

    Beheshti, Afshin

    2002-04-01

    Separations were performed on double stranded DNA (dsDNA) using electrophoresis. Electrophoresis is the steady transport of particles under the influence of an external electric field. Double stranded DNA fragments ranging in length from 200 base pairs (bp) to 194,000 bp (0.34 nm = 1 bp) were electrophoresed at agarose gel concentrations T = 0.4%--1.5%. The electric field was varied from 0.62 V/cm to 6.21 V/cm. A wide range of electric fields and gel concentrations were used to study the usefulness of a new interpolation equation, 1mL =1mL-( 1mL-1 ms)e-L/g , where mL,ms , and g are independent free fitting parameters. The long length mobility limit is interpreted as mL , the short length mobility limit is ms , and g is the crossover between the long length limit and the short length limit. This exponential relation fit very well (chi2 ≥ 0.999) when there are two smooth transitions observed in the "reptation plots" (plotting 3mL/m∘ vs. L) (J. Rousseau, G. Drouin, and G. W. Slater, Phys Rev Lett. 1997, 79, 1945--1948). Fits deviate from the data when three different slopes were observed in the reptation plots. Reptation plots were used to determine a phase diagram for dsDNA migration regimes. The phase diagrams define different regions where mechanisms for molecular transport affect the migration of dsDNA in agarose gels during electrophoresis. The parameters from the equation have also been interpreted to provide a physical description of the structure of the agarose gel by calculating the pore sizes. The relations between the values for the pore sizes and the phase diagrams are interpreted to better understand the migration of the DNA through agarose gels.

  8. Isoelectric focusing in agarose gel for detection of oligoclonal bands in cerebrospinal and other biological fluids.

    PubMed

    Csako, Gyorgy

    2012-01-01

    Isoelectric focusing (IEF) coupled with immunodetection (immunofixation or immunoblotting) has become the leading technique for the detection and study of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) and also is increasingly used in other body fluids such as the tear and serum. Limited commercial availability of precast agarose IEF gels for research and a need for customization prompted reporting a detailed general protocol for the preparation and casting of agarose IEF gel along with sample, control, and isoelectric point marker preparation and carrying out the focusing itself for CSF OCBs. However, the method is readily adaptable to the use of other body fluid specimens and, possibly, research specimens such as culture fluids as well.

  9. Two-dimensional agarose gel electrophoresis for analysis of DNA replication.

    PubMed

    Villwock, Sandra K; Aparicio, Oscar M

    2014-01-01

    The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.

  10. Confirmation of soybean plastid rRNAs by formaldehyde denaturing agarose gel electrophoresis.

    PubMed

    Zhu, Y Q; Zheng, Y; Chen, H B; Huang, L Q

    2014-10-27

    Owing to their prokaryotic origin, plastid rRNAs are mainly 23s/16s/5s rRNAs. We present a novel plant RNA isolation method in this paper. Also, not only the eukaryotic 28s (26s, 25s)/18s rRNAs but the prokaryotic 26s/23s rRNAs as well were demonstrated in a single sample for the first time by formaldehyde denaturing agarose gel electrophoresis.

  11. Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction.

    PubMed

    Kurien, B T; Kaufman, K M; Harley, J B; Scofield, R H

    2001-09-15

    A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.

  12. Selective bacterial patterning using the submerged properties of microbeads on agarose gel.

    PubMed

    Park, Sung Jun; Bae, Hyeoni; Ko, Seong Young; Min, Jung-Joon; Park, Jong-Oh; Park, Sukho

    2013-10-01

    We proposed a new bacteria patterning method on the restricted region of microbeads, using the submerged property of polystyrene microbeads on various concentrations of agarose gel. Moreover, we fabricated a bacterial microrobot using attenuated Salmonella typhimurium through the new patterning methods. We controlled the submerged degree of polystyrene microbeads through the regulation of the hardness of the agarose gel. The polystyrene microbeads on agarose gel were transferred onto a poly-dimethylsiloxane (PDMS) surface for easy manipulation of the microbeads. Then, we treated the polystyrene microbeads on the PDMS surface with antibacterial adherent factors, such as O2 plasma and bovine serum albumin (BSA). The Salmonella typhimurium was attached to the entire surface of the untreated polystyrene microbeads, whereas Salmonella typhimurium were only attached to the restricted surface region of the treated polystyrene microbeads through the proposed patterning method. The bacteria-attached microbeads gain motility by the propulsion of the attached bacteria, and the selective-bacteria-attached microbeads showed enhanced motility. Compared with whole-bacteria-attached polystyrene microbeads (1.74 ± 1.62 μm/s), the selective bacteria-attached polystyrene microbeads, using O2 plasma and BSA, showed 9.18 ± 1.88 μm/s and 14.65 ± 8.66 μm/s faster moving velocities, respectively. Through the results, we expected that the proposed patterning methodology of microbeads could contribute to the development of biomedical bacterial microrobots.

  13. Capillary blotting of glycosaminoglycans on nitrocellulose membranes after agarose-gel electrophoresis separation.

    PubMed

    Volpi, Nicola; Maccari, Francesca

    2009-01-01

    A method for the blotting and immobilizing of several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by cationic detergent after their separation by conventional agarose gel electrophoresis is illustrated. This new approach to the study of glycosaminoglycans (GAGs) utilizes the capacity of agarose gel electrophoresis to separate single species of polysaccharides from mixtures and the membrane technology for further preparative and analytical uses.Nitrocellulose membranes are derivatized with the cationic detergent cetylpyridinium chloride and mixtures of GAGs are capillary blotted after their separation in agarose gel electrophoresis. Single purified species of variously sulfated polysaccharides are transferred on derivatized membranes with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining). This enables a lower amount limit of detection of 0.1 microg. Nonsulfated polyanions, for example hyaluronic acid, may also be transferred to membranes with a limit of detection of approximately 0.1-0.5 microg after irreversible or reversible staining. The membranes may be stained with reversible staining and the same lanes are used for immunological detection or other applications.

  14. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

    PubMed

    Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40(T), was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

  15. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.

    PubMed

    Sanderson, Brian A; Araki, Naoko; Lilley, Jennifer L; Guerrero, Gilberto; Lewis, L Kevin

    2014-06-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution.

  16. Methods for determining agent concentration profiles in agarose gel during convection-enhanced delivery.

    PubMed

    Sindhwani, Nikhil; Ivanchenko, Oleksandr; Lueshen, Eric; Prem, Komal; Linninger, Andreas A

    2011-03-01

    Convection-enhanced delivery (CED) is a promising technique to deliver large molecular weight drugs to the human brain for treatment of Parkinson's, Alzheimer's, or brain tumors. Researchers have used agarose gels to study mechanisms of agent transport in soft tissues like brain due to its similar mechanical and transport properties. However, inexpensive quantitative techniques to precisely measure achieved agent distribution in agarose gel phantoms during CED are missing. Such precise measurements of concentration distribution are needed to optimize drug delivery. An optical experimental method to accurately quantify agent concentration in agarose is presented. A novel geometry correction algorithm is used to determine real concentrations from observable light intensities captured by a digital camera. We demonstrate the technique in dye infusion experiments that provide cylindrical and spherical distributions when infusing with porous membrane and conventional single-port catheters, respectively. This optical method incorporates important parameters, such as optimum camera exposure, captured camera intensity calibration, and use of collimated light source for maximum precision. We compare experimental results with numerical solutions to the convection diffusion equation. The solutions of convection-diffusion equations in the cylindrical and spherical domains were found to match the experimental data obtained by geometry correction algorithm.

  17. Agarose gels

    NASA Astrophysics Data System (ADS)

    2016-11-01

    Discovered in 17th-century Japan, agar is a jelly-like substance obtained by boiling algae, and it is widely used as a gelling agent for desserts in Japanese, Indian, Philippine and Vietnamese cuisine.

  18. Cytoplasmic polyhedrosis virus classification by electropherotype; validation by serological analyses and agarose gel electrophoresis.

    PubMed

    Mertens, P P; Crook, N E; Rubinstein, R; Pedley, S; Payne, C C

    1989-01-01

    Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris-HCl/Tris-glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris-acetate-EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates.

  19. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution.

    PubMed

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-07-15

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.

  20. Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis.

    PubMed

    Issa, Samah M A; Schulz, Benjamin L; Packer, Nicolle H; Karlsson, Niclas G

    2011-12-01

    Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.

  1. Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan.

    PubMed

    Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K

    2011-10-01

    Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5-500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 μg (for polyacrylamide) to 2.5 μg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150-kDa HA standard.

  2. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis.

    PubMed

    Vetcher, Alexandre A; Srinivasan, Srimeenakshi; Vetcher, Ivan A; Abramov, Semen M; Kozlov, Mikhail; Baughman, Ray H; Levene, Stephen D

    2006-08-28

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  3. Stabilization of thin-layer agarose gels after isoelectric focusing with polyacrylamide enables reverse imidazole-zinc staining and facilitates two-dimensional gel electrophoresis.

    PubMed

    Hellman, Jukka

    2008-09-01

    Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated.

  4. Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan.

    PubMed

    Cowman, Mary K; Chen, Cherry C; Pandya, Monika; Yuan, Han; Ramkishun, Dianne; LoBello, Jaclyn; Bhilocha, Shardul; Russell-Puleri, Sparkle; Skendaj, Eraldi; Mijovic, Jovan; Jing, Wei

    2011-10-01

    The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100°C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.

  5. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel.

    PubMed

    Zhang, Dongwei; Das, Diganta B; Rielly, Chris D

    2014-02-01

    A set of laboratory experiments has been carried out to determine if micro-needles (MNs) can enhance penetration depths of high-speed micro-particles delivered by a type of gene gun. The micro-particles were fired into a model target material, agarose gel, which was prepared to mimic the viscoelastic properties of porcine skin. The agarose gel was chosen as a model target as it can be prepared as a homogeneous and transparent medium with controllable and reproducible properties allowing accurate determination of penetration depths. Insertions of various MNs into gels have been analysed to show that the length of the holes increases with an increase in the agarose concentration. The penetration depths of micro-particle were analysed in relation to a number of variables, namely the operating pressure, the particle size, the size of a mesh used for particle separation and the MN dimensions. The results suggest that the penetration depths increase with an increase of the mesh pore size, because of the passage of large agglomerates. As these particles seem to damage the target surface, then smaller mesh sizes are recommended; here, a mesh with a pore size of 178 μm was used for the majority of the experiments. The operating pressure provides a positive effect on the penetration depth, that is it increases as pressure is increased. Further, as expected, an application of MNs maximises the micro-particle penetration depth. The maximum penetration depth is found to increase as the lengths of the MNs increase, for example it is found to be 1272 ± 42, 1009 ± 49 and 656 ± 85 μm at 4.5 bar pressure for spherical micro-particles of 18 ± 7 μm diameter when we used MNs of 1500, 1200 and 750 μm length, respectively.

  6. Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels

    SciTech Connect

    Sutherland, B.M.; Shih, A.G.

    1983-02-15

    We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weight and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

  7. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis.

    PubMed

    Schvartzman, J B; Martínez-Robles, M L; Hernández, P; Krimer, D B

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

  8. Tailor-made cell patterning using a near-infrared-responsive composite gel composed of agarose and carbon nanotubes.

    PubMed

    Koga, Haruka; Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi; Nakazawa, Kohji

    2013-03-01

    Micropatterning is useful for regulating culture environments. We developed a highly efficient near-infrared-(NIR)-responsive gel and established a new technique that enables cell patterning by NIR irradiation. As a new culture substratum, we designed a tissue culture plate that was coated with a composite gel composed of agarose and carbon nanotubes (CNTs). A culture plate coated with agarose only showed no response to NIR irradiation. In contrast, NIR laser irradiation induced heat generation by CNTs; this permitted local solation of the CNT/agarose gel, and consequently, selective cell-adhesive regions were exposed on the tissue culture plate. The solation area was controlled by the NIR intensity, magnification of the object lens and CNT concentration in the gel. Furthermore, we formed circular patterns of HeLa cells and linear patterns of 3T3 cells on the same culture plate through selective and stepwise NIR irradiation of the CNT/agarose gel, and we also demonstrated that individual 3T3 cells migrated along a linear path formed on the CNT/agarose gel by NIR irradiation. These results indicate that our technique is useful for tailor-made cell patterning of stepwise and/or complex cell patterns, which has various biological applications such as stepwise co-culture and the study of cell migration.

  9. An agarose gel-based neurosphere culture system leads to enrichment of neuronal lineage cells in vitro.

    PubMed

    Park, Kyuhee; Nam, Yeonju; Choi, Yongmun

    2015-05-01

    Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into β-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.

  10. A New Organic Dye-Based Staining for The Detection of Plant DNA in Agarose Gels.

    PubMed

    Sönmezoğlu, Özlem Ateş; Özkay, Kerime

    2015-01-01

    Ethidium bromide (EtBr) is used to stain DNA in agarose gel electrophoresis, but this dye is mutagenic and carcinogenic. We investigated N-719, which is a visible, reliable and organic Ruthenium-based dye, and five fluorescent alternatives for staining plant DNA. For prestaining and poststaining, N-719, GelRed, and SYBR Safe stained both DNA and PCR product bands as clearly as EtBr. SYBR Green I, methylene blue, and crystal violet were effective for poststaining only. The organic dye N-719 stained DNA bands as sensitively and as clearly as EtBr. Consequently, organic dyes can be used as alternatives to EtBr in plant biotechnology studies.

  11. Assaying cooperativity of protein-DNA interactions using agarose gel electrophoresis.

    PubMed

    Williams, Tanya L; Levy, Daniel L

    2013-01-01

    DNA-binding proteins play essential roles in many cellular processes. Understanding on a molecular level how these proteins interact with their cognate sequences can provide important functional insights. Here, we describe a band shift assay in agarose gel to assess the mode of protein binding to a DNA molecule containing multiple protein-binding sites. The basis for the assay is that protein-DNA complexes display retarded gel electrophoresis mobility, due to their increased molecular weight relative to free DNA. The degree of retardation is higher with increasing numbers of bound protein molecules, thereby allowing resolution of complexes with differing protein-DNA stoichiometries. The DNA is radiolabeled to allow for visualization of both unbound DNA and all the different DNA-protein complexes. We present a quantitative analysis to determine whether protein binding to multiple sites within the same DNA molecule is independent or cooperative.

  12. Agarose gel-coated LPG based on two sensing mechanisms for relative humidity measurement.

    PubMed

    Miao, Yinping; Zhang, Kaikiang; Yuam, Yujie; Liu, Bo; Zhang, Hao; Liu, Yan; Yao, Jianquan

    2013-01-01

    A relative humidity (RH) sensor based on long-period grating (LPG) with different responses is proposed by utilizing agarose gel as the sensitive cladding film. The spectral characteristic is discussed as the ambient humidity level ranges from 25% to 95% RH. Since increment of RH will result in volume expansion and refractive index increment of the agarose gel, the LPG is sensitive to applied strain and ambient refractive index; both the resonance wavelength and coupling intensity present particular responses to RH within two different RH ranges (25%-65% RH and 65%-96% RH). The coupling intensity decreases within a lower RH range while it increases throughout a higher RH range. The resonance wavelength is sensitive to the higher RH levels, and the highest sensitivity reaches 114.7 pm/% RH, and shares the same RH turning point with coupling intensity response. From a practical perspective, the proposed RH sensor would find its potential applications in high humidity level, temperature-independent RH sensing and multiparameter sensing based on wavelength/power hybrid demodulation and even static RH alarm for automatic monitoring of a particular RH value owing to the nonmonotonic RH dependence of the transmission power within the whole tested RH range.

  13. Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations.

    PubMed

    Volpi, Nicola; Buzzega, Dania

    2012-01-01

    The adulteration of raw heparin (Hep) with a synthetic oversulfated chondroitin sulfate (OSCS) not found in nature produced in 2007-2008 a global crisis giving rise to the development of additional, new and specific methods for its quality assurance and purity. In this study, a simple and sensitive agarose-gel electrophoresis method has been developed for the visualization of OSCS in Hep samples along with other natural glycosaminoglycans possibly present as "process-related impurities", in particular dermatan sulfate (DS) and chondroitin sulfate (CS). Agarose-gel electrophoresis under non-conventional conditions is able to separate OSCS from Hep with its two components, the slow-moving and fast-moving species, DS and CS by performing separation for 15 h (overnight) and under high voltage (100 mA, ∼200 V). Densitometric scanning enabled us to calculate a limit of detection of ∼0.5 μg OSCS with a linear behaviour from 0.1 to 5 μg, comparable to CS/DS. Contaminated samples from Hep manufacturers were analyzed and quantitative data were found comparable to previous studies. Due to its capacity to process many samples in a single run and to the equipment commonly available in laboratories, this analytical method would be suitable for the identification and quantification of contamination by other polysaccharides, in particular OSCS and DS, within Hep preparations and formulations.

  14. Detection of genotoxic insult as DNA strand breaks in fish blood cells by agarose gel electrophoresis

    SciTech Connect

    Theodorakis, C.W. ); D'Surney, S.J. . Dept. of Biology); Shugart, L.R. . Environmental Sciences Division)

    1994-07-01

    DNA, isolated from the blood cells of bluegill sunfish (Lepomis macrochirus) exposed in the lab to bedded sediment collected from a site contaminated with genotoxic compounds (i.e., PAHs, PCBs, and heavy metals), was examined for strand breakage by agarose gel electrophoresis. Before electrophoresis the blood cells were embedded in agarose plugs and incubated with proteinase. After electrophoresis under both neutral (pH 7) or alkaline (pH 12) conditions, the median molecular length (MML) of the DNA distributed in the gel was determined. These quantitative measures were used to estimate the difference in the number of double- and single-strand breaks between DNA preparations. Both types of strand breakage were found to be greater in fish exposed to sediment contaminated with genotoxic compounds as compared to nonexposed fish. A statistically significant correlation was demonstrated between the MML value obtained by the electrophoretic assay reported here and the F value (measure of DNA double-strandedness) obtained by the alkaline unwinding assay.

  15. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins.

    PubMed

    McCudden, Christopher R; Mathews, Stephanie P; Hainsworth, Shirley A; Chapman, John F; Hammett-Stabler, Catherine A; Willis, Monte S; Grenache, David G

    2008-03-01

    The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

  16. Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

  17. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    SciTech Connect

    Kedersha, N.L.; Hill, D.F.; Kronquist, K.E.; Rome, L.H.

    1986-01-01

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis, the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from difference populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Stripped CVs exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetyl-choline esterase, and an M/sub r/ 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from /sup 125/I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.

  18. Visualization of DNA in agarose gels as migrating colored bands: Applications to laboratory techniques

    SciTech Connect

    Adkins, S.; Burmeister, M.

    1994-09-01

    We have developed a method to visualize DNA without the use of ethidium bromide and UV radiation. Anionic dyes (colored anion) have long been used in the detection of pharmaceutical amines via ion pairing, here we show that cationic dyes may be used to detect DNA. In gel electrophoresis in which DNA is traveling toward the positive electrode and a cationic dye is traveling toward the negative electrode, we expect ion pairing of the DNA and the dye as they meet in the gel. The dye should bind to the anionic DNA. If the DNA is not completely neutralized by the dye, it should continue to migrate. Ethidium bromide, which is believed to stain DNA primarily by intercalation between bases, exhibits the fluorescence through its cation and also may bind to DNA, to some extent, through ionic pairing. We observed that DNA forms colored bands during electrophoresis in standard agarose gels when a cationic dye is present in the gel and running buffer. DNA in amounts equal to or greater than 80 ng is seen as a discrete migrating colored band in ambient room lighting. Colored bands may be transferred to nitrocellulose by vacuum transfer in room temperature gel dryer, Xeroxed, fixed with NaOH and dye removed with dilute detergent. Also, isolation of DNA bands from preparative gels may be accomplished without the typical use of ethidium bromide and UV radiation which are known to alter DNA and pose hazards to laboratory personnel. We are presently investigating the general utility of using dyes to visualize DNA for various laboratory techniques.

  19. Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis.

    PubMed

    Holt, Ian J; Kazak, Lawrence; Reyes, Aurelio; Wood, Stuart R

    2016-01-01

    Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the "bootlace mechanism of mitochondrial DNA replication," in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.

  20. Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis.

    PubMed

    Holmberg, Mats; Nollen, Ellen A A

    2013-01-01

    The accumulation of specific aggregation-prone proteins during aging is thought to be involved in several diseases, most notably Alzheimer's and Parkinson's disease as well as polyglutamine expansion disorders such as Huntington's disease. Caenorhabditis elegans disease models with transgenic expression of fluorescently tagged aggregation-prone proteins have been used to screen for genetic modifiers of aggregation. To establish the role of modifying factors in the generation of aggregation intermediates, a method has been developed using native agarose gel electrophoresis (NAGE) that enables parallel screening of aggregation patterns of fluorescently labeled aggregation-prone proteins. Together with microscopy-based genetic screens this method can be used to identify modifiers of protein aggregation and characterize their molecular function. Although described here for analyzing aggregates in C. elegans, NAGE can be adjusted for use in other model organisms as well as for cultured cells.

  1. Effects of DS-modified agarose gels on neurite extension in 3D scaffold through mechanisms other than changing the pore radius of the gels.

    PubMed

    Peng, Jin; Pan, Qian; Zhang, Wei; Yang, Hao; Zhou, Xue; Jiang, Hua

    2014-07-01

    Dermatan sulfate is widely distributed as glycosaminoglycan side chains of proteoglycans, which are the main components of glial scar and inhibit neurite regeneration after nerve injury. However its role in the inhibiting process is not clear. Understanding neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study used agarose gels modified with dermatan sulfate as the three-dimensional culture scaffold. We explored structure-function relationship between the three-dimensional scaffold and neurite extension and examined the role of dermatan sulfate on neurite extension in the three-dimensional scaffold. A range of agarose concentrations was used to generate varied gel physical structures and the corresponding neurite extension of embryonic day (E9) chick dorsal root ganglia was examined. We measured gel stiffness and gel pore size to determine whether dermatan sulfate changed the gels' conformation. As gel concentration increased, neurite length and gel pore size decreased, and gel stiffness increased. At 1.00 and 1.25% (wt/vol) concentrations, dermatan sulfates both immobilized with agarose gels and dissolved in culture medium inhibit neurite extension. While at 1.50 and 1.75% (wt/vol) concentrations, only immobilized dermatan sulfate worked. Immobilized dermatan sulfate could modify molecular shape of agarose gels, decrease gel pore size statistically, but did not influence gel stiffness. We have proved that the decrease of gel pore size is insufficient to inhibit neurite extension. These results indicate that dermatan sulfate inhibits neurite extension not through forming a mechanical barrier. Maybe its interaction with neuron membrane is the key factor in neurite extension.

  2. Isoelectric focusing of human von Willebrand factor in urea-agarose gels

    SciTech Connect

    Fulcher, C.A.; Ruggeri, Z.M.; Zimmerman, T.S.

    1983-02-01

    An analytical technique has been developed for the isoelectric focusing (IEF) of plasma von Willebrand factor (vWF) in agarose gels containing urea. Under these conditions, vWF freely enters the gel and focuses without artifact. The focused vWF is visualized by staining fixed gels with /sup 125/I-labeled affinity-purified heterologous antibody. Utilizing a pH gradient of 5.0-6.5, normal vWF in plasma or purified preparations focuses into at least three bands with apparent isoelectric points (pI) between pH 5.7 and 5.9. A reproducible difference in the IEF pattern of vWF has been established between normal plasmas and those of individuals with variant von Willebrand's disease (vWd) type IIA and type IIB. In type IIA, vWF has a distinctly lower pI than normal. This difference may be related to the presence of smaller vWF multimers in IIA plasma because forms of vWF of corresponding size contained in normal cryoprecipitate supernatant have a similar pI. Type IIB von Willebrand factor has a pI intermediate between normal and IIA. Neuraminidase treatment of plasma samples before IEF results in an increase in pI in normal, type IIA, and type IIB vWF. The data suggest that none of the 16 type IIA and 9 IIB plasmas studied here contain significantly decreased amounts of sialic acid.

  3. Measurement of DNA damage using agarose gel electrophoresis and electronic imaging

    SciTech Connect

    Sutherland, J.C.; Bergman, A.M.; Chen, Chun-Zhang; Monteleone, D.C.; Trunk, J.; Sutherland, B.M.

    1988-01-01

    Damage done to DNA by ultraviolet (uv) light, gamma rays and other carcinogens can be quantified using agarose gel electrophororesis. Agents that either produce strand breaks directly or that produce lesions that can be enzymatically or chemically converted to strand breaks can be studied. The method requires: (1) accurate measurement of the disribution of mass of DNA as a function of the distance of migration in the gel, (2) determination of the dispersion function of the electrophoresis system and (3) calculation of weighted averages of these functions by a computer. Less than 50 ng of DNA are required and the DNA need not be labeled with a radioactive tracer. Hence, the damage and repair of DNA in non-dividing cells and intact organisms---including humans---can be studied. Initial applications have focused on the quantitation of cyclobutyl pyrimidine dimers in the DNA of uv irradiated human skin. The sensitivity of lesion detection is increased by unidirectional pulsed field electrophoresis and other methods that separate longer DNA molecules. Replacing photographic detection of ethidium fluorescence by electronic imaging increases the accuracy of the measurement and the speed of data analysis. Quantitative electronic imaging of gel fluorescence offers advantages over photography in other areas of molecular biology, medicine and biotechnology. 26 refs., 5 figs.

  4. Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis.

    PubMed

    Yokoyama, Eiji; Kishida, Kazunori; Uchimura, Masako; Ichinohe, Sadato

    2006-06-01

    Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.

  5. Electrophoresis of /sup 35/S-labeled proteoglycans of polyacrylamide-agarose composite gels and their visualization by fluorography

    SciTech Connect

    Carney, S.L.; Bayliss, M.T.; Collier, J.M.; Muir, H.

    1986-01-01

    Techniques for the electrophoresis of /sup 35/S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms have been developed. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

  6. Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.

    PubMed Central

    Roninson, I B

    1983-01-01

    A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines. Images PMID:6310499

  7. Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

    2003-11-01

    Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

  8. Influence of pinning effects on the electrochemical formation of silver patterns in agarose-containing sols and gels.

    PubMed

    Pasquale, M A; Saracco, G P; Marchiano, S L; Arvia, A J

    2005-11-03

    The formation of silver patterns via electrolysis from aqueous silver sulfate + x% w/v agarose sol and gel media, with and without supporting electrolyte, in a quasi-two-dimensional (2D) cylindrical cell at room temperature, is utilized as a reference system to investigate the complexity of pinning effects. From pattern morphology and electrochemical data, both delocalized and localized pinning in the bulk dominate the drift of the growth front, depending on the concentration of agarose in the heterogeneous media. Delocalized pinning results from mobile, small agarose aggregates at the growth front and from their accumulation by the front drift. For gels, localized pinning comes from their own percolated structure. A depinning/pinning transition is observed in going from sols to gels. The relative contribution of diffusion and advection in mass-transport-controlled silver electrodeposition depends on the plating bath composition. On the other hand, silver ion attachment to the cathode appears to be interfered with by some screening caused by weakly adsorbed, mobile agarose aggregates at the metal surface without slowing down the rate of the electron-transfer step at the cathode. Their relative contribution of a delocalized, localized pinning and screening effect to a great extent determines the morphology and transition in the growth mode of silver patterns in both media. The analysis of charge and current transients and the corresponding silver pattern morphologies for open and dense radial patterns is made. Results are qualitatively simulated with a novel, rather simple cellular automaton algorithm.

  9. A shortcut organic dye-based staining method for the detection of DNA both in agarose and polyacrylamide gel electrophoresis.

    PubMed

    Cong, Weitao; Chen, Mao; Zhu, Zhongxin; Liu, Zhiguo; Nan, Jia; Ye, Weijian; Ni, Maowei; Zhao, Ting; Jin, Litai

    2013-02-21

    In this study, we describe a brief, sensitive and safe organic dye-based staining method for the visualization of DNA both in agarose and polyacrylamide gels by using Victoria Pure Blue BO (VPBBO). Down to 0.8-1.6 ng of λ DNA/HindIII markers in agarose gels and 0.4-0.8 ng of pUC18 DNA/Mspl markers in polyacrylamide gels can be successfully detected within 15 and 10 min by the new developed technique, respectively. Moreover, the mechanism of the VPBBO staining was investigated and further confirmed by electrospray ionization mass spectrometry (ESI-MS) and molecular docking. The results indicated that the interaction between VPBBO and DNA is mainly due to groove binding.

  10. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    PubMed

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  11. Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

    PubMed Central

    Serwer, Philip; Wright, Elena T.

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

  12. Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

    PubMed

    Serwer, Philip; Wright, Elena T

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging.

  13. Model creation of moving redox reaction boundary in agarose gel electrophoresis by traditional potassium permanganate method.

    PubMed

    Xie, Hai-Yang; Liu, Qian; Li, Jia-Hao; Fan, Liu-Yin; Cao, Cheng-Xi

    2013-02-21

    A novel moving redox reaction boundary (MRRB) model was developed for studying electrophoretic behaviors of analytes involving redox reaction on the principle of moving reaction boundary (MRB). Traditional potassium permanganate method was used to create the boundary model in agarose gel electrophoresis because of the rapid reaction rate associated with MnO(4)(-) ions and Fe(2+) ions. MRB velocity equation was proposed to describe the general functional relationship between velocity of moving redox reaction boundary (V(MRRB)) and concentration of reactant, and can be extrapolated to similar MRB techniques. Parameters affecting the redox reaction boundary were investigated in detail. Under the selected conditions, good linear relationship between boundary movement distance and time were obtained. The potential application of MRRB in electromigration redox reaction titration was performed in two different concentration levels. The precision of the V(MRRB) was studied and the relative standard deviations were below 8.1%, illustrating the good repeatability achieved in this experiment. The proposed MRRB model enriches the MRB theory and also provides a feasible realization of manual control of redox reaction process in electrophoretic analysis.

  14. High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.

    PubMed

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.

  15. Analysis of Telomere-Homologous DNA with Different Conformations Using 2D Agarose Electrophoresis and In-Gel Hybridization.

    PubMed

    Zhang, Zepeng; Hu, Qian; Zhao, Yong

    2017-01-01

    In mammalian cells, in addition to double-stranded telomeric DNA at chromosome ends, extra telomere-homologous DNA is present that adopts different conformations, including single-stranded G- or C-rich DNA, extrachromosomal circular DNA (T-circle), and telomeric complex (T-complex) with an unidentified structure. The formation of such telomere-homologous DNA is closely related to telomeric DNA metabolism and chromosome end protection by telomeres. Conventional agarose gel electrophoresis is unable to separate DNA based on conformation. Here, we introduce the method of two-dimensional (2D) agarose electrophoresis in combination with in-gel native/denatured hybridization to determine different conformations formed by telomere-homologous DNA.

  16. A method for direct application of human plasmin on a dithiothreitol-containing agarose stacking gel system.

    PubMed

    Choi, Nack-Shick; Chung, Dong-Min; Yoon, Kab-Seog; Maeng, Pil Jae; Kim, Seung-Ho

    2005-11-30

    A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1 %) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.

  17. Measurement of Ferric Ion Diffusion Coefficient in Fricke-Infused Agarose Gel From MR Image Intensity Changes

    DTIC Science & Technology

    2007-11-02

    Coefficient in Fricke-Infused Agarose Gel From MR Image Intensity Changes Contract Number Grant Number Program Element Number Author( s ) Project Number Task...Number Work Unit Number Performing Organization Name( s ) and Address(es) Institutes of Biomedical Engineering and Radiological Sciences National...Yang Ming University Pei-Tou Taipei, Taiwan, R.O.C. Performing Organization Report Number Sponsoring/Monitoring Agency Name( s ) and Address(es) US

  18. Analysis of DNA structures from eukaryotic cells by two-dimensional native-native DNA agarose gel electrophoresis.

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The neutral-neutral two-dimensional agarose gel technique is mainly used to determine the chromosomal positions where DNA replication starts, but it is also applied to visualize replication fork progression and breakage as well as intermediates in DNA recombination. Here we provide a step-by-step protocol to analyze the fairly underrepresented and fragile replication intermediates in yeast chromosomal DNA. The technique can also be adapted to analyze replication intermediates in chromosomal DNA of higher eukaryotic organisms.

  19. Rheological and mechanical behavior of polyacrylamide hydrogels chemically crosslinked with allyl agarose for two-dimensional gel electrophoresis.

    PubMed

    Suriano, R; Griffini, G; Chiari, M; Levi, M; Turri, S

    2014-02-01

    Two-dimensional (2-D) gel electrophoresis currently represents one of the most standard techniques for protein separation. In addition to the most commonly employed polyacrylamide crosslinked hydrogels, acrylamide-agarose copolymers have been proposed as promising systems for separation matrices in 2-D electrophoresis, because of the good resolution of both high and low molecular mass proteins made possible by careful control and optimization of the hydrogel pore structure. As a matter of fact, a thorough understanding of the nature of the hydrogel pore structure as well as of the parameters by which it is influenced is crucial for the design of hydrogel systems with optimal sieving properties. In this work, a series of acrylamide-based hydrogels covalently crosslinked with different concentrations of allyl agarose (0.2-1%) is prepared and characterized by creep-recovery measurements, dynamic rheology and tensile tests, in the attempt to gain a clearer understanding of structure-property relationships in crosslinked polyacrylamide-based hydrogels. The rheological and mechanical properties of crosslinked acrylamide-agarose hydrogels are found to be greatly affected by crosslinker concentration. Dynamic rheological tests show that hydrogels with a percentage of allyl agarose between 0.2% and 0.6% have a low density of elastically effective crosslinks, explaining the good separation of high molecular mass proteins in 2-D gel electrophoresis. Over the same range of crosslinker concentration, creep-recovery measurements reveal the presence of non-permanent crosslinks in the hydrogel network that justifies the good resolution of low molecular mass proteins as well. In tensile tests, the hydrogel crosslinked with 0.4% of allyl agarose exhibits the best results in terms of mechanical strength and toughness. Our results show how the control of the viscoelastic and the mechanical properties of these materials allow the design of mechanically stable hydrogels with improved

  20. Photothermal Microneedle Etching: Improved Three-Dimensional Microfabrication Method for Agarose Gel for Topographical Control of Cultured Cell Communities

    NASA Astrophysics Data System (ADS)

    Moriguchi, Hiroyuki; Yasuda, Kenji

    2006-08-01

    We have developed a new three-dimensional (3D) microfabrication method for agarose gel, photothermal microneedle etching (PTMNE), by means of an improved photothermal spot heating using a focused 1064 nm laser beam for melting a portion of the agarose layer at the tip of the microneedle, where a photoabsorbent chromium layer is coated to be heated. The advantage of this method is that it allows the 3D control of the melting topography within the thick agarose layer with a 2 μm resolution, whereas conventional photothermal etching can enable only two-dimensional (2D) control on the surface of the chip. By this method, we can form the spheroid clusters of particular cells from isolated single cells without any physical contact with other cells in other chambers, which is important for measuring the community effect of the cell group from isolated single cells. When we set single cancer cells in microchambers of 100 μm in diameter, formed in a 50-μm-thick agarose layer, we observed that they grew, divided, and formed spheroid clusters of cells in each microchamber. The result indicates the potential of this method to be a fundamental technique in the research of multicellular spherical clusters of cells for checking the community effect of cells in 3D structures, such as the permeabilities of chemicals and substrates into the cluster, which is complementary to conventional 2D dish cultivation and can contribute to the cell-based screening of drugs.

  1. Electroelution of nucleic acids from polyacrylamide gels: a custom-made, agarose-based electroeluter.

    PubMed

    Fadouloglou, Vasiliki E

    2013-06-01

    Polyacrylamide electrophoresis is routinely used for small-scale preparative and analytical separations. The incomparably high-resolution separations achieved by this technique, however, have not been widely exploited to the large-scale preparative isolation of biological molecules from contaminants, mainly because of difficulties in the recovery of the desired molecule from the gel matrix. Electroelution is an effective procedure applied for this purpose. However, commercially available, high-cost electroeluters are required for achieving high recovery yields. Here, we describe a custom-made electroeluter that combines low-cost, high-recovery yields, short times of electroelution, and convenience in the manipulation of sensitive samples.

  2. Molecular imprinting in sol-gel matrix.

    PubMed

    Gupta, Radha; Kumar, Ashok

    2008-01-01

    Molecular imprinting is a newly developed methodology which provides molecular assemblies of desired structures and properties and is being increasingly used for several applications such as in separation processes, microreactors, immunoassays and antibody mimics, catalysis, artificial enzymes, biosensor recognition elements and bio- and chemo-sensors. The ambient processing conditions and versatility of the sol-gel process makes sol-gel glassy matrix suitable for molecular imprinting. The progress of sol-gel based molecular imprinted polymers (MIPs) for various applications can be seen from the growing number of publications. The main focus of the review is molecular imprinting in sol-gel matrix and applications of molecular imprinted sol-gel derived materials for the development of sensors. Combining sol-gel process with molecular imprinting enables to procure the sensors with greater sensitivity and selectivity necessary for sensing applications. The merits, problems, challenges and factors affecting molecular imprinting in sol-gel matrix have been discussed. Considerable attention has been drawn on recent developments like use of organically modified silane precursors (ORMOSILS) for the synthesis of hybrid molecular imprinted polymers (HMIPs) and applying surface sol-gel process for molecular imprinting. The development of molecular imprinted sol-gel nanotubes for biochemical separation and bio-imprinting is a new advancement and is under progress. Templated xerogels and molecularly imprinted sol-gel films provide a good platform for various sensor applications.

  3. Biomimetic materials processing: Implementation of molecular imprinting and study of biomineralization through the development of an agarose gel assay

    NASA Astrophysics Data System (ADS)

    Boggavarapu, Sajiv

    agarose gel matrix for studying inhibition and growth as influenced by various organic molecule functionalities. The gel mineralization assay is a novel approach in which quantitative and qualitative data could be generated in a high throughput fashion to determine organic molecule mediation of calcium based crystal growth. Such methods provide an approach for eventually providing control in development of synthetic biocomposites with customized materials properties.

  4. Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose.

    PubMed

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Ujihara, Hiromi; Koike, Tohru

    2009-08-01

    We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).

  5. A new approach for calibration of laser ablation inductively coupled plasma mass spectrometry using thin layers of spiked agarose gels as references.

    PubMed

    Stärk, H-J; Wennrich, Rainer

    2011-02-01

    Calibration of analytical methods using laser ablation for sample introduction is often problematic. The availability of matrix-adapted standard materials is a crucial factor in the analysis of biological samples in particular. In this work a method for preparation of thin-film references for LA-ICP-MS is presented which is inexpensive, relatively simple and generally practicable. Aqueous solutions of agarose spiked with defined amounts of the analytes were cast on a carrier and then dried. When the thin-film references were characterized the average thickness of the films was 0.03 mm in the centre of the film and the relative standard deviation was 8%. Nebulization ICP-MS analysis after acid digestion of the agarose film was used to investigate the effectiveness of the spiking procedure. Recovery of the spiked elements was frequently in the range 90-110% (for rare earth elements 97-102%). Laser ablation ICP-MS analysis was used to investigate the distribution of the spiked elements in the film. When the laser was scanned across the gel the measured intensities were not constant, but had a peak-shaped profile with a flat top. Use of this flat-top region for analytical purposes, after its characterization by laser ablation ICP-MS, is proposed. Analysis of cell cultures was carried out by direct laser ablation-ICP-MS with the calibration method described. The results were in accordance with values previously achieved by nebulization ICP-MS.

  6. Rapid drug susceptibility test of Mycobacterium tuberculosis using microscopic time-lapse imaging in an agarose matrix.

    PubMed

    Choi, Jungil; Yoo, Jungheon; Kim, Ki-Jung; Kim, Eun-Geun; Park, Kyung Ock; Kim, Hyejin; Kim, Haeun; Jung, Hyunju; Kim, Taeyoung; Choi, Myungjin; Kim, Hee Chan; Ryoo, Sungweon; Jung, Yong-Gyun; Kwon, Sunghoon

    2016-03-01

    Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients.

  7. A crystallization technique for obtaining large protein crystals with increased mechanical stability using agarose gel combined with a stirring technique

    NASA Astrophysics Data System (ADS)

    Maruyama, Mihoko; Hayashi, Yuki; Yoshikawa, Hiroshi Y.; Okada, Shino; Koizumi, Haruhiko; Tachibana, Masaru; Sugiyama, Shigeru; Adachi, Hiroaki; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Takano, Kazufumi; Murakami, Satoshi; Yoshimura, Masashi; Mori, Yusuke

    2016-10-01

    We developed a protein crystallization technique using a 0.0-2.0 w/v% agarose gel solution combined with a stirring technique for the purpose of controlling the crystal number in the gelled solutions. To confirm the stirring effect in the gelled solution, we investigated the nucleation probability and growth rate of the crystals produced using this method. The stirring operation by a rotary shaker affected the behavior of protein molecules in the gelled solution, and both a significant decrease in the nucleation rate and an enhancement of the crystal growth rate were achieved by the method. As a result, we concluded that the proposed technique, the stirring technique in a gel solution, was effective for generating protein crystals of sufficient and increased mechanical stability.

  8. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.

    PubMed

    Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrüggen, Susanne

    2012-12-01

    Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein.

  9. Characterization of a heterogeneous chicken plasma protein, HEF, by analytical isotachophoresis in agarose gel.

    PubMed

    Nicolaisen, E M

    1985-02-22

    Chicken plasma contains proteins that associate with immunoglobulin. One of these proteins enhances the titre of haemagglutinating alloantibodies, and it was therefore named HEF, haemagglutination enhancing factor. A purified HEF preparation mixed with ampholytes splits into four bands in analytical agarose isotachophoresis. One of the HEF bands can be separated from two others with beta-alanine as discrete spacer. The separated HEF populations differ in molecular size and in their ability to enhance agglutination.

  10. Ag@AgI, core@shell structure in agarose matrix as hybrid: synthesis, characterization, and antimicrobial activity.

    PubMed

    Ghosh, Somnath; Saraswathi, A; Indi, S S; Hoti, S L; Vasan, H N

    2012-06-05

    A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV-visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria.

  11. An enzyme-entrapped agarose gel for visualization of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system.

    PubMed

    Tanaka, Kazuhisa; Shoji, Atushi; Sugawara, Masao

    2015-01-01

    An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 μmol L(-1) for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler's Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated.

  12. A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

    PubMed Central

    Glazer, A N; Peck, K; Mathies, R A

    1990-01-01

    The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble. Images PMID:2339125

  13. A stable double-stranded DNA-ethidium homodimer complex: Application to picogram fluorescence detection of DNA in agarose gels

    SciTech Connect

    Glazer, A.N.; Mathies, R.A. Lawrence Berkeley Laboratory, CA ); Peck, K. )

    1990-05-01

    The complex between double-stranded DNA and ethidium homodimer (5,5{prime}-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridinium) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the performed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

  14. Agarose gel as biomaterial or scaffold for implantation surgery: characterization, histological and histomorphometric study on soft tissue response.

    PubMed

    Varoni, Elena; Tschon, Matilde; Palazzo, Barbara; Nitti, Paola; Martini, Lucia; Rimondini, Lia

    2012-01-01

    Maxillofacial, orthopedic, oral, and plastic surgery require materials for tissue augmentation, guided regeneration, and tissue engineering approaches. In this study, the aim was to develop and characterize a new extrudable hydrogel, based on agarose gel (AG; 1.5% wt) and to evaluate the local effects after subcutaneous implantation in comparison with collagen and hyaluronic acid. AG chemical-physical properties were ascertained through Fourier transform infrared (FT-IR) spectroscopy and rheological analysis. In vivo subcutaneous implants were performed, and histological and histomorphometric evaluations were done at 1, 4, 12, and 16 weeks. FT-IR confirmed that spectroscopic properties were the same for the baseline agarose and rheological characterization established that AG is a weak hydrogel. Subcutaneous AG implants induced new vessels and fibrous tissue formation rich in neutrophils; the capsule thickness around AG increased until the 12th week but remained thinner than those around hyaluronic acid and collagen. At 16 weeks, the thickness of the capsule significantly decreased around all materials. This study confirmed that 1.5% wt AG possesses some of the most important features of the ideal biocompatible material: safety, effectiveness, costless, and easily obtained with specific chemical and geometrical characters; the AG can represent a finely controllable and biodegradable polymeric system for cells and drug delivery applications.

  15. Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: analysis example using Mycobacterium tuberculosis.

    PubMed

    Wada, Takayuki; Maeda, Shinji

    2013-04-01

    As one genotyping method for Mycobacterium tuberculosis, variable number of tandem repeats (VNTR) is a promising tool to trace the undefined transmission of tuberculosis, but it often requires large equipment such as a genetic analyzer for DNA fragment analysis or CE system to conduct systematic analyses. For convenient genotyping at low cost in laboratories, we designed a multiplex PCR system that is applicable to agarose gel electrophoresis using fluorescent PCR primers. For tuberculosis genotyping by VNTR, the copy quantities of minisatellite DNA must be determined in more than 12 loci. The system can halve laborious electrophoresis processes by presenting an image of two VNTR amplicons on a single lane. No expensive equipment is necessary for this method. Therefore, it is useful even in developing countries.

  16. A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel.

    PubMed

    Cao, Hui-Ling; Sun, Li-Hua; Li, Jian; Tang, Lin; Lu, Hui-Meng; Guo, Yun-Zhu; He, Jin; Liu, Yong-Ming; Xie, Xu-Zhuo; Shen, He-Fang; Zhang, Chen-Yan; Guo, Wei-Hong; Huang, Lin-Jun; Shang, Peng; He, Jian-Hua; Yin, Da-Chuan

    2013-10-01

    High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

  17. Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography.

    PubMed

    Yi, C F; Gosiewska, A; Burtis, D; Geesin, J

    2001-04-01

    The currently available methods for the detection of proteases in tissue sections are characterized by limited substrate specificity and low sensitivity and are also cumbersome. We have developed a novel in situ zymography method that uses a synthetic substrate conjugated to a fluorescent tag for detection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulting in an approximately 100-fold increase in the fluorescent signal. In order to minimize the diffusion of the fluorescent tag, the substrate is incorporated into 1% agarose prior to overlaying onto the tissue section. This method retains the morphological details of the tissue section, is highly sensitive and specific for the designated peptide sequence, and provides information regarding the functional status of the enzyme. Thus, this method could be used for detection and monitoring of enzymatic activity in tissue sections for a variety of applications.

  18. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    ERIC Educational Resources Information Center

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  19. DNA electrophoresis in agarose gels: Effects of electric field and gel concentration on the exponential dependence of reciprocal mobility on DNA length

    NASA Astrophysics Data System (ADS)

    Beheshti, Afshin; van Winkle, David; Randolph, Rill

    2002-03-01

    Electrophoresis was performed on double stranded DNA fragments ranging in length from 200 bp to 48502 bp at agarose gel concentrations T = 0.5% - 1.5% and electric fields E = 0.71 V/cm to 5 V/cm. A wide range of electric fields and gel concentrations were used to find what range of conditions work with the new interpolation equation, 1/μ(L) = 1/μl - (1/μl - 1/μ_s)e^-L/γ. The equation fit extremely well (\\chi^2 >= 0.999) to data with E = 2.5 V/cm to 5 V/cm and for lower fields (E < 2.5 V/cm) at low gel concentrations (T = 0.5% and 0.7%). This exponential relation seemed to hold when there is a smooth transition from the Ogston sieving regime to the reptation regime when looking at the “reptation plots” (plotting 3μL/μo vs. L) (Rousseau, J., Drouin, G., and Slater, G. W., Phys Rev Lett. 1997, 79, 1945-1948). For separations of single-stranded DNA in polyacrylamide, similar reptation plots have a region with a negative slope between the Ogston sieving regime and the reptation regime which has been interpreted as the signature of entropic trapping. When separating double-stranded DNA in agarose it was observed that fits deviate from the data when three different slopes are observed in the reptation plots. Failure of the simple exponential relationship between reciprocal mobility and DNA length appears to be the consequence of entropic trapping.

  20. DNA electrophoresis in agarose gels: effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length.

    PubMed

    Rill, Randolph L; Beheshti, Afshin; Van Winkle, David H

    2002-08-01

    Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.

  1. The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis.

    PubMed Central

    Schvartzman, J B; Martínez-Robles, M L; Hernández, P

    1993-01-01

    Initiation of DNA replication in higher eukaryotes is still a matter of controversy. Some evidence suggests it occurs at specific sites. Data obtained using two-dimensional (2D) agarose gel electrophoresis, however, led to the notion that it may occur at random in broad zones. This hypothesis is primarily based on the observation that several contiguous DNA fragments generate a mixture of the so-called 'bubble' and 'simple Y' patterns in Neutral/neutral 2D gels. The interpretation that this mixture of hybridisation patterns is indicative for random initiation of DNA synthesis relies on the assumption that replicative intermediates (RIs) containing an internal bubble where initiation occurred at different relative positions, generate comigrating signals. The latter, however, is still to be proven. We investigated this problem by analysing together, in the same 2D gel, populations of pBR322 RIs that were digested with different restriction endonucleases that cut the monomer only once at different locations. DNA synthesis begins at a specific site in pBR322 and progresses in a uni-directional manner. Thus, the main difference between these sets of RIs was the relative position of the origin. The results obtained clearly showed that populations of RIs containing an internal bubble where initiation occurred at different relative positions do not generate signals that co-migrate all-the-way in 2D gels. Despite this observation, however, our results support the notion that random initiation is indeed responsible for the peculiar 'bubble' signal observed in the case of several metazoan eukaryotes. Images PMID:8265365

  2. Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

    PubMed Central

    Martín-Parras, L; Lucas, I; Martínez-Robles, M L; Hernández, P; Krimer, D B; Hyrien, O; Schvartzman, J B

    1998-01-01

    Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells. PMID:9649629

  3. Beverage-Agarose Gel Electrophoresis: An Inquiry-Based Laboratory Exercise with Virtual Adaptation

    ERIC Educational Resources Information Center

    Cunningham, Steven C.; McNear, Brad; Pearlman, Rebecca S.; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As…

  4. Evaluation of the friction coefficient, the radial stress, and the damage work during needle insertions into agarose gels.

    PubMed

    Urrea, Fabián A; Casanova, Fernando; Orozco, Gustavo A; García, José J

    2016-03-01

    Agarose hydrogels have been extensively used as a phantom material to mimic the mechanical behavior of soft biological tissues, e.g. in studies aimed to analyze needle insertions into the organs producing tissue damage. To better predict the radial stress and damage during needle insertions, this study was aimed to determine the friction coefficient between the material of commercial catheters and hydrogels. The friction coefficient, the tissue damage and the radial stress were evaluated at 0.2, 1.8, and 10mm/s velocities for 28, 30, and 32 gauge needles of outer diameters equal to 0.36, 0.31, and 0.23mm, respectively. Force measurements during needle insertions and retractions on agarose gel samples were used to analyze damage and radial stress. The static friction coefficient (0.295±0.056) was significantly higher than the dynamic (0.255±0.086). The static and dynamic friction coefficients were significantly smaller for the 0.2mm/s velocity compared to those for the other two velocities, and there was no significant difference between the friction coefficients for 1.8 and 10mm/s. Radial stress averages were 131.2±54.1, 248.3±64.2, and 804.9±164.3Pa for the insertion velocity of 0.2, 1.8, and 10mm/s, respectively. The radial stress presented a tendency to increase at higher insertion velocities and needle size, which is consistent with other studies. However, the damage work did not show to be a good predictor of tissue damage, which appears to be due to simplifications in the analytical model. Differently to other approaches, the method proposed here based on radial stress may be extended in future studies to quantity tissue damage in vivo along the entire needle track.

  5. Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

    PubMed

    Sun, Guowei; Xiao, Jinzhou; Lu, Man; Wang, Hongming; Chen, Xiaobing; Yu, Yongxin; Pan, Yingjie; Wang, Yongjie

    2015-01-01

    The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

  6. A robust new strategy for high-molecular-weight proteome research: a 2-hydroxyethyl agarose/polyacrylamide gel enhanced separation and ZnO-PMMA nanobeads assisted identification.

    PubMed

    Shen, Wenwen; Shen, Chengpin; Xiong, Huanming; Lu, Haojie; Yang, Pengyuan

    2010-09-15

    A new mass spectrometry based analysis strategy has been established here for high-molecular-weight (HMW) proteome research. First, a 2-hydroxyethyl agarose/polyacrylamide (HEAG/PAM) electrophoresis gel was designed for the first time to realize an easy-handling separation method with high spatial resolution for HMW proteins, good reproducibility and mass spectrometry-compatible silver staining. Second, ZnO-polymethyl methacrylate (ZnO-PMMA) nanobeads were applied here for enriching and desalting the peptides from the HMW proteins. Third, the peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with the presence of the ZnO-PMMA nanobeads, and their MS signals were enhanced markedly. The success rate of identification for HMW proteins was significantly increased due to high enriching efficiency and salt tolerance capability as well as signal enhancing capability of the ZnO-PMMA nanobeads. We believe that this analysis strategy will inspire and accelerate the HMW proteome studies.

  7. Effect of gel structure of matrix orientation in pulsed alternating electric fields

    SciTech Connect

    Stellwagen, N.C.; Stellwagen, J.

    1993-12-31

    Four polymeric gels with different structures, LE agarose, HEEO agarose, beta-carrageenan, and polyacrylamide, were studied by transient electric birefringence to determine the importance of various structural features on the orientation of the gels in pulsed alternating electric fields. The birefrigence relaxation times observed for agarose gels in low voltage electric fields suggest that long fibers and/or domains, ranging up to tens of microns in size, are oriented by the electric field. The sign of the birefringence reverses when the direction of the electric field is reversed, suggesting that the oriented domains change their direction of orientation from parallel to perpendicular (or vice versa) when the polarity of the electric field is reversed. These anamalous orientation effects are observed with both types of agarose gels, but not with beta-carrageenan or polyacrylamide gels, suggesting that the alternating D,L galactose residues in the agarose backbone are responsible for the anomalies.

  8. Microscale mechanisms of agarose-induced disruption of collagen remodeling.

    PubMed

    Ulrich, Theresa A; Lee, Tae Geol; Shon, Hyun Kyong; Moon, Dae Won; Kumar, Sanjay

    2011-08-01

    Cells are strongly influenced by the local structure and mechanics of the extracellular matrix (ECM). We recently showed that adding agarose to soft collagen ECMs can mechanically stiffen these hydrogels by two orders of magnitude while limiting 3D cell motility, which we speculated might derive from agarose-mediated inhibition of collagen fiber deformation and remodeling. Here, we directly address this hypothesis by investigating the effects of agarose on cell-collagen interactions at the microscale. Addition of agarose progressively restricts cell spreading, reduces stress fiber and focal adhesion assembly, and inhibits macroscopic gel compaction. While time-of-flight secondary ion mass spectrometry and scanning electron microscopy fail to reveal agarose-induced alterations in collagen ligand presentation, the latter modality shows that agarose strongly impairs cell-directed assembly of large collagen bundles. Agarose-mediated inhibition of cell spreading and cytoarchitecture can be rescued by β-agarase digestion or by covalently crosslinking the matrix with glutaraldehyde. Based on these results, we argue that cell spreading and motility on collagen requires local matrix stiffening, which can be achieved via cell-mediated fiber remodeling or by chemically crosslinking the fibers. These findings provide new mechanistic insights into the regulatory function of agarose and bear general implications for cell adhesion and motility in fibrous ECMs.

  9. Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

    SciTech Connect

    Pagratis, N.; Revel, H.R. )

    1990-07-01

    Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.

  10. Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis.

    PubMed

    Jeiranian, H Arthur; Schalow, Brandy J; Courcelle, Justin

    2010-12-21

    Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

  11. Plasma protein electrophoresis in birds: comparison of a semiautomated agarose gel system with an automated capillary system.

    PubMed

    Roman, Yannick; Bomsel-Demontoy, Marie-Claude; Levrier, Julie; Chaste-Duvernoy, Daniel; Saint Jalme, Michel

    2013-06-01

    Plasma agarose gel electrophoresis (AGE) is recognized as a very reliable diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary zone electrophoresis (CZE) have emerged in human laboratory medicine but have never been investigated in birds. To investigate the use of CZE in birds and to compare it with AGE, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 racing pigeons (Columba livia) were analyzed by both AGE and CZE. For the 3 species studied, values determined by AGE and CZE were well correlated for albumin and beta and gamma fractions whereas other values differed significantly. Values for alpha-3 fraction in the rooster, alpha-1 fraction in the black kite, and alpha fractions in the pigeon obtained by AGE were very well correlated with the prealbumin fraction values obtained by CZE. Repeatability and reproducibility appeared higher with CZE than with AGE. Although the interpretation of CZE electrophoresis patterns seems to produce results similar to those obtained with AGE, some proteins present in the alpha fraction measured with AGE migrated to the prealbumin fraction found with CZE. Although CZE requires the use of specific reference intervals and a much higher sample volume, this method has many advantages when compared with AGE, including better repeatability and reproducibility and higher analysis output.

  12. Lambda light chain myeloma with co-migrating paraprotein at beta region on agarose gel electrophoresis: a case report.

    PubMed

    Siti Sarah, M; Nor Aini, U; Nurismah, M I; Hafiza, A; Khalidah, M; Mokhtar, A B; Das, S

    2014-01-01

    Paraproteinemia is one of the diagnostic features of multiple myeloma. A commonly used method is the detection of paraprotein by agarose gel electrophoresis (AGE) followed by by immunofixation electrophoresis (IFE) to confirm monoclonality. Due to their smaller size, immunoglobulin A (IgA) and light chain only paraproteins may appear at the beta or even alpha 2 protein fractions. Here, we discuss a case report of a 47-year-old man who presented with pathological fracture of third thoracic (T3) vertebra. Serum protein electrophoresis (SPE) was initially reported as no paraprotein detected. However, a bone biopsy was reported to show plasma cell proliferation with light chain restriction. A repeat sample for protein electrophoresis together with IFE revealed lambda light chain paraprotein co-migrating at the beta region. The beta band plus paraprotein was quantitated as 4.3 g/L (7.0%), which was within normal limits of the beta protein fraction. Hence, it has to be remembered that if the SPE is negative, it does not necessarily mean that the paraprotein is absent in cases which are highly suspicious.

  13. Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis.

    PubMed

    Dziedziejko, Violetta; Safranow, Krzysztof; Slowik-Zylka, Dorota; Machoy-Mokrzynska, Anna; Millo, Barbara; Machoy, Zygmunt; Chlubek, Dariusz

    2009-03-01

    Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.

  14. Comparison of three methods of DNA extraction in endocervical specimens for Chlamydia trachomatis infection by spectrophotometry, agarose gel, and PCR.

    PubMed

    Jenab, Anahita; Roghanian, Rasoul; Golbang, Naser; Golbang, Pouran; Chamani-Tabriz, Leili

    2010-06-01

    Chlamydia trachomatis is the major cause of sexually transmitted disease in the world. The aim of this study was to determine the best method of DNA extraction for detecting C. trachomatis by polymerase chain reaction (PCR) in sexually active women (n = 80) attending Shahid Beheshti Hospital in Isfahan, Iran. Endocervical swabs were collected from 80 women, 22 of whom were asymptomatic and 58 symptomatic. Three different DNA extraction methods were used in this study (phenol-chlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by beta-globin primers (PCO4, GH20). The DNA cryptic plasmid was selected as the target for C. trachomatis and samples were examined by PCR using specific KL1 and KL2 primers. It was shown that DNA extraction by boiling was the most sensitive with the highest yield of DNA. Of the 80 samples, 17 (21.25%) showed positivity for C. trachomatis by PCR. The highest rate of C. trachomatis infection was found in the group aged between 35 and 45 years old and those who used withdrawal or an intrauterine device as methods of contraception. It was demonstrated that DNA extraction by boiling was the least expensive and a very rapid method that gave the highest DNA yield. The infection rate in the sexually active women, including symptomatic and asymptomatic, was 21.25%, with a presumably high prevalence compared with other studies done in this field.

  15. A simple approach for preparation of affinity matrices: Simultaneous purification and reversible immobilization of a streptavidin mutein to agarose matrix

    PubMed Central

    Wu, Sau-Ching; Wang, Chris; Hansen, Dave; Wong, Sui-Lam

    2017-01-01

    SAVSBPM18 is an engineered streptavidin for affinity purification of both biotinylated biomolecules and recombinant proteins tagged with streptavidin binding peptide (SBP) tags. To develop a user-friendly approach for the preparation of the SAVSBPM18-based affinity matrices, a designer fusion protein containing SAVSBPM18 and a galactose binding domain was engineered. The galactose binding domain derived from the earthworm lectin EW29 was genetically modified to eliminate a proteolytic cleavage site located at the beginning of the domain. This domain was fused to the C-terminal end of SAVSBPM18. It allows the SAVSBPM18 fusions to bind reversibly to agarose and can serve as an affinity handle for purification of the fusion. Fluorescently labeled SAVSBPM18 fusions were found to be stably immobilized on Sepharose 6B-CL. The enhanced immobilization capability of the fusion to the agarose beads results from the avidity effect mediated by the tetrameric nature of SAVSBPM18. This approach allows the consolidation of purification and immobilization of SAVSBPM18 fusions to Sepharose 6B-CL in one step for affinity matrix preparation. The resulting affinity matrix has been successfully applied to purify both SBP tagged β-lactamase and biotinylated proteins. No significant reduction in binding capacity of the column was observed for at least six months. PMID:28220817

  16. A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis

    SciTech Connect

    Dewey, W.C.; Thompson, L.L.; Trinh, M.L.; Latz, D.L. |; Ward, J.F.

    1994-10-01

    A charge-coupled-device camera system was coupled to a personal computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (<400 nm) from the agarose gel, and by using neutral density filters to prevent saturation of the camera, the fluorescence intensity is linearly related to the amount of DNA varying from {approximately} 0.03 {mu}g in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) to {approximately} 4 {mu}g (where ethidium bromide staining saturates). The percentage DNA released from the plug and distribution in the lane (with 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of radioactivity of the same DNA labeled with [{sup 3}H]dThd. However, scattering of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for {approximately} 7% of the fluorescence from the plug tailing out {approximately} 10 mm beyond the first 1 mm in the lane. In addition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA migration stops. 31 refs., 7 figs.

  17. Effect of Polyethylene Glycol on Properties and Drug Encapsulation-Release Performance of Biodegradable/Cytocompatible Agarose-Polyethylene Glycol-Polycaprolactone Amphiphilic Co-Network Gels.

    PubMed

    Chandel, Arvind K Singh; Kumar, Chinta Uday; Jewrajka, Suresh K

    2016-02-10

    We synthesized agarose-polycaprolactone (Agr-PCL) bicomponent and Agr-polyethylene glycol-PCL (Agr-PEG-PCL) tricomponent amphiphilic co-network (APCN) gels by the sequential nucleophilic substitution reaction between amine-functionalized Agr and activated halide terminated PCL or PCL-b-PEG-b-PCL copolymer for the sustained and localized delivery of hydrophilic and hydrophobic drugs. The biodegradability of the APCNs was confirmed using lipase and by hydrolytic degradation. These APCN gels displayed good cytocompatibility and blood compatibility. Importantly, these APCN gels exhibited remarkably high drug loading capacity coupled with sustained and triggered release of both hydrophilic and hydrophobic drugs. PEG in the APCNs lowered the degree of phase separation and enhanced the mechanical property of the APCN gels. The drug loading capacity and the release kinetics were also strongly influenced by the presence of PEG, the nature of release medium, and the nature of the drug. Particularly, PEG in the APCN gels significantly enhanced the 5-fluorouracil loading capacity and lowered its release rate and burst release. Release kinetics of highly water-soluble gemcitabine hydrochloride and hydrophobic prednisolone acetate depended on the extent of water swelling of the APCN gels. Cytocompatibility/blood compatibility and pH and enzyme-triggered degradation together with sustained release of drugs show great promise for the use of these APCN gels in localized drug delivery and tissue engineering applications.

  18. Directed, strong, and reversible immobilization of proteins tagged with a β-trefoil lectin domain: a simple method to immobilize biomolecules on plain agarose matrixes.

    PubMed

    López-Gallego, Fernando; Acebrón, Ivan; Mancheño, Jose Miguel; Raja, Sebastian; Lillo, M Pilar; Guisán Seijas, Jose Manuel

    2012-03-21

    A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus . Such a domain (LSL(150)), recently described as a novel fusion tag, is based on a β-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL(150) to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the LSL(150)-agarose interaction. Furthermore, this latter interaction is temperature dependent since it is 4-fold stronger when the immobilization takes place at 25 °C than when it does at 4 °C. The strongest lectin-agarose interaction is also quite stable under a survey of different conditions such as high temperatures (up to 60 °C) or high organic solvent concentrations (up to 60% of acetonitrile). Notably, the use of cross-linked agarose would endow the system with more robustness due to its better mechanical properties compared to the noncross-linked one. The stability of the LSL(150)-agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL(150) lectin domain exhibits interesting structural features as an immobilization domain that makes it suitable to reversibly immobilize industrially relevant enzymes in very simple carriers as agarose.

  19. Comparison between a second generation automated multicapillary electrophoresis system with an automated agarose gel electrophoresis system for the detection of M-components.

    PubMed

    Larsson, Anders; Hansson, Lars-Olof

    2008-01-01

    During the last decade, capillary electrophoresis (CE) has emerged as an interesting alternative to traditional analysis of serum, plasma and urine proteins by agarose gel electrophoresis. Initially there was a considerable difference in resolution between the two methods but the quality of CE has improved significantly. We thus wanted to evaluate a second generation of automated multicapillary instruments (Capillarys, Sebia, Paris, France) and the high resolution (HR) buffer for serum or plasma protein analysis with an automated agarose gel electrophoresis system for the detection of M-components. The comparison between the two systems was performed with patients samples with and without M-components. The comparison included 76 serum samples with M-components > 1 g/L. There was a total agreement between the two methods for detection of these M-components. When studying samples containing oligoclonal bands/small M-components, there were differences between the two systems. The capillary electrophoresis system detected a slightly higher number of samples with oligoclonal bands but the two systems found oligoclonal bands in different samples. When looking at resolution, the agarose gel electrophoresis system yielded a slightly better resolution in the alpha and beta regions, but it required an experienced interpreter to be able to benefit from the increased resolution. The capillary electrophoresis has shorter turn-around times and bar-code reader that allows positive sample identification. The Capillarys in combination with HR buffer gives better resolution of the alpha and beta regions than the same instrument with the beta1-beta2+ buffer or the Paragon CZE2000 (Beckman) which was the first generation of capillary electrophoresis systems.

  20. Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium.

    PubMed

    Fujihara, M; Comizzoli, P; Wildt, D E; Songsasen, N

    2012-12-01

    Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.

  1. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  2. High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

    PubMed

    Giori, Luca; Tricomi, Flavia Marcella; Zatelli, Andrea; Roura, Xavier; Paltrinieri, Saverio

    2011-07-01

    The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

  3. Fabrication of Self-Healable and Patternable Polypyrrole/Agarose Hybrid Hydrogels for Smart Bioelectrodes.

    PubMed

    Park, Nokyoung; Chae, Seung Chul; Kim, Il Tae; Hur, Jaehyun

    2016-02-01

    We present a new class of electrically conductive, mechanically moldable, and thermally self-healable hybrid hydrogels. The hybrid gels consist of polypyrrole and agarose as the conductive component and self-healable matrix, respectively. By using the appropriate oxidizing agent under conditions of mild temperature, the polymerization of pyrrole occurred along the three-dimensional network of the agarose hydrogel matrix. In contrast to most commercially available hydrogels, the physical crosslinking of agarose gel allows for reversible gelation in the case of our hybrid gel, which could be manipulated by temperature variation, which controls the electrical on/off behavior of the hybrid gel electrode. Exploiting this property, we fabricated a hybrid conductive hydrogel electrode which also self-heals thermally. The novel composite material we report here will be useful for many technological and biological applications, especially in reactive biomimetic functions and devices, artificial muscles, smart membranes, smart full organic batteries, and artificial chemical synapses.

  4. A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

    PubMed

    Serwer, P; Hayes, S J; Moreno, E T; Park, C Y

    1992-09-15

    Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Encapsulation of chondrocytes in high-stiffness agarose microenvironments for in vitro modeling of osteoarthritis mechanotransduction.

    PubMed

    Jutila, Aaron A; Zignego, Donald L; Schell, William J; June, Ronald K

    2015-05-01

    In articular cartilage, chondrocytes reside within a gel-like pericellular matrix (PCM). This matrix provides a mechanical link through which joint loads are transmitted to chondrocytes. The stiffness of the PCM decreases in the most common degenerative joint disease, osteoarthritis. To develop a system for modeling the stiffness of both the healthy and osteoarthritic PCM, we determined the concentration-stiffness relationships for agarose. We extended these results to encapsulate chondrocytes in agarose of physiological stiffness. Finally, we assessed the relevance of stiffness for chondrocyte mechanotransduction by examining the biological response to mechanical loading for cells encapsulated in low- and high-stiffness gels. We achieved agarose equilibrium stiffness values as large as 51.3 kPa. At 4.0% agarose, we found equilibrium moduli of 34.3 ± 1.65 kPa, and at 4.5% agarose, we found equilibrium moduli of 35.7 ± 0.95 kPa. Cyclical tests found complex moduli of ~100-300 kPa. Viability was >96% for all studies. We observed distinct metabolomic responses in >500 functional small molecules describing changes in cell physiology, between primary human chondrocytes encapsulated in 2.0 and 4.5% agarose indicating that the gel stiffness affects cellular mechanotransduction. These data demonstrate both the feasibility of modeling the chondrocyte pericellular matrix stiffness and the importance of the physiological pericellular stiffness for understanding chondrocyte mechanotransduction.

  6. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

    SciTech Connect

    Mathew, M.K.; Smith, C.L.; Cantor, C.R. )

    1988-12-27

    Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1,500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.

  7. Sol/Gel Processing Techniques for Glass Matrix Composites.

    DTIC Science & Technology

    1987-11-01

    development of a general technique (i.e., Pyrex is less susceptible to devitrification than SiO2 or TiO2 -SiO 2 ). In addition. the properties of these sol / gel ...of a sol / gel process for SIC 2 and SiO2 - TiO2 - together with a data base for their densification - are prerequisite to the successful fabrication of...S~%ad~ 5~ ~ ~ *~~~~;:>;::L-; 1: ’*~~’~ ’S. AFWL-TN-86-59 AFWL-TN- 86-59 00 SOL / GEL PROCESSING TECHNIQUES FOR GLASS MATRIX COMPOSITES 0) C. G

  8. In-gel expression and in situ immobilization of proteins for generation of three dimensional protein arrays in a hydrogel matrix.

    PubMed

    Byun, Ju-Young; Lee, Kyung-Ho; Lee, Ka-Young; Kim, Min-Gon; Kim, Dong-Myung

    2013-03-07

    A method has been developed for the direct conversion of DNA arrays into three dimensional protein arrays on a hydrogel matrix. An agarose gel embedded with bacterial protein synthesis machinery was used as the DNA-programmable expression gel matrix for the in situ translation of genes on a DNA array. Upon incubation of the expression gel matrix cast on a DNA array, protein synthesis took place at the interface of the two surfaces and the cell-free synthesized proteins were deposited on the gel matrix surrounding the corresponding DNA spots. Diffusional dilution of the expressed proteins was minimized by modifying the agarose with Ni-NTA moieties. This procedure resulted in the generation of localized protein spots with confined radii. The developed approach not only simplifies the procedures typically used for the preparation of protein arrays but it also provides conditions for the loading of higher amounts of proteins on the array while retaining their structural integrity and functionality over extended time periods.

  9. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    NASA Astrophysics Data System (ADS)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  10. New polymer gel dosimeters consisting of less toxic monomers with radiation-crosslinked gel matrix

    NASA Astrophysics Data System (ADS)

    Hiroki, A.; Yamashita, S.; Sato, Y.; Nagasawa, N.; Taguchi, M.

    2013-06-01

    New polymer gel dosimeters consisting of less toxic methacrylate-type monomers such as 2-hydroxymethyl methacrylate (HEMA) and polyethylene glycol 400 dimethacrylate (9G) with hydroxypropyl cellulose (HPC) gel were prepared. The HPC gels were obtained by using a radiation-induced crosslinking technique to be applied in a matrix instead of a gelatin, which is conventionally used in earlier dosimeters, for the polymer gel dosimeters. The prepared polymer gel dosimeters showed cloudiness by exposing to 60Co γ-ray, in which the cloudiness increased with the dose up to 10 Gy. At the same dose, the increase in the cloudiness appeared with increasing concentration of 9G. As a result of the absorbance measurement, it was found that the dose response depended on the composition ratio between HEMA and 9G.

  11. The mechanical microenvironment of high concentration agarose for applying deformation to primary chondrocytes.

    PubMed

    Zignego, Donald L; Jutila, Aaron A; Gelbke, Martin K; Gannon, Daniel M; June, Ronald K

    2014-06-27

    Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ~25-200kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72h in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ~1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

  12. Temporal effect of inertial cavitation with and without microbubbles on surface deformation of agarose S gel in the presence of 1-MHz focused ultrasound.

    PubMed

    Tomita, Y; Matsuura, T; Kodama, T

    2015-01-01

    Sonoporation has the potential to deliver extraneous molecules into a target tissue non-invasively. There have been numerous investigations of cell membrane permeabilization induced by microbubbles, but very few studies have been carried out to investigate sonoporation by inertial cavitation, especially from a temporal perspective. In the present paper, we show the temporal variations in nano/micro-pit formations following the collapse of inertial cavitation bubbles, with and without Sonazoid® microbubbles. Using agarose S gel as a target material, erosion experiments were conducted in the presence of 1-MHz focused ultrasound applied for various exposure times, Tex (0.002-60 s). Conventional microscopy was used to measure temporal variations in micrometer-scale pit numbers, and atomic force microscopy utilized to detect surface roughness on a nanometer scale. The results demonstrated that nanometer-scale erosion was predominantly caused by Sonazoid® microbubbles and C4F10 gas bubbles for 0.002 s

  13. Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

    PubMed

    Villagomez, Rodrigo; Hatti-Kaul, Rajni; Sterner, Olov; Almanza, Giovanna; Linares-Pastén, Javier A

    2015-01-01

    The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

  14. Native agarose gel electrophoresis and electroelution: A fast and cost-effective method to separate the small and large hepatitis B capsids.

    PubMed

    Yoon, Kam Yee; Tan, Wen Siang; Tey, Beng Ti; Lee, Khai Wooi; Ho, Kok Lian

    2013-01-01

    Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

  15. Immobilization of Aspergillus oryzae β-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose.

    PubMed

    Guerrero, Cecilia; Vera, Carlos; Serna, Nestor; Illanes, Andrés

    2017-02-07

    Aspergillus oryzae β-galactosidase was immobilized in monofunctional glyoxyl-agarose and heterofunctional supports (amino-glyoxyl, carboxy-glyoxyl and chelate-glyoxyl agarose), for obtaining highly active and stable catalysts for lactulose synthesis. Specific activities of the amino-glyoxyl agarose, carboxy-glyoxyl agarose and chelate-glyoxyl agarose derivatives were 3676, 430 and 454IU/g biocatalyst with half-life values at 50°C of 247, 100 and 100h respectively. Specific activities of 3490, 2559 and 1060IU/g were obtained for fine, standard and macro agarose respectively. High immobilization yield (39.4%) and specific activity of 7700IU/g was obtained with amino-glyoxyl-agarose as support. The highest yields of lactulose synthesis were obtained with monofunctional glyoxyl-agarose. Selectivity of lactulose synthesis was influenced by the support functionalization: glyoxyl-agarose and amino-glyoxyl-agarose derivatives retained the selectivity of the free enzyme, while selectivity with the carboxy-glyoxyl-agarose and chelate-glyoxyl-agarose derivatives was reduced, favoring the synthesis of transgalactosylated oligosaccharides over lactulose.

  16. Application of urea-agarose gel electrophoresis to select non-redundant 16S rRNAs for taxonomic studies: palladium(II) removal bacteria.

    PubMed

    Assunção, Ana; Costa, Maria Clara; Carlier, Jorge Dias

    2016-03-01

    The 16S ribosomal RNA (rRNA) gene has been the most commonly used sequence to characterize bacterial communities. The classical approach to obtain gene sequences to study bacterial diversity implies cloning amplicons, selecting clones, and Sanger sequencing cloned fragments. A more recent approach is direct sequencing of millions of genes using massive parallel technologies, allowing a large-scale biodiversity analysis of many samples simultaneously. However, currently, this technique is still expensive when applied to few samples; therefore, the classical approach is still used. Recently, we found a community able to remove 50 mg/L Pd(II). In this work, aiming to identify the bacteria potentially involved in Pd(II) removal, the separation of urea/heat-denatured DNA fragments by urea-agarose gel electrophoresis was applied for the first time to select 16S rRNA-cloned amplicons for taxonomic studies. The major raise in the percentage of bacteria belonging to genus Clostridium sensu stricto from undetected to 21 and 41 %, respectively, for cultures without, with 5 and 50 mg/L Pd(II) accompanying Pd(II) removal point to this taxa as a potential key agent for the bio-recovery of this metal. Despite sulfate-reducing bacteria were not detected, the hypothesis of Pd(II) removal by activity of these bacteria cannot be ruled out because a slight decrease of sulfate concentration of the medium was verified and the formation of PbS precipitates seems to occur. This work also contributes with knowledge about suitable partial 16S rRNA gene regions for taxonomic studies and shows that unidirectional sequencing is enough when Sanger sequencing cloned 16S rRNA genes for taxonomic studies to genus level.

  17. Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering.

    PubMed

    Yamada, Yuji; Hozumi, Kentaro; Aso, Akihiro; Hotta, Atsushi; Toma, Kazunori; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

    2012-06-01

    Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering.

  18. Pulse Field Gel Electrophoresis

    PubMed Central

    Sharma-Kuinkel, Batu K.; Rude, Thomas H.; Fowler, Vance G.

    2015-01-01

    Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments. PMID:25682374

  19. Pulse Field Gel Electrophoresis.

    PubMed

    Sharma-Kuinkel, Batu K; Rude, Thomas H; Fowler, Vance G

    2016-01-01

    Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

  20. A surfactant-based, regularly arrayed nanostructure gel matrix for migration of small molecules.

    PubMed

    Kato, Masaru; Suwanai, Yusuke; Shimojima, Atsushi; Santa, Tomofumi

    2012-11-01

    The preparation of nanometer-scale pores, or nanopores, has become easy because of the progress in nanotechnology. Surfactants are promising materials for the preparation of nanostructures containing nanopores, because surfactants form many different phase structures, including cubic, micellar, and lamellar structures. We prepared a gel matrix with a cubic structure from a commercially available surfactant, polyoxyethylene(50) lauryl ether (C12EO50, Adekatol LA-50). This gel matrix had regularly arrayed nanopores between the packed spherical micelles. We used the gel to separate biomolecules by means of slab gel electrophoresis. The gel was applicable to migration of amino acids and peptides; however, larger molecules, such as proteins and single-walled carbon nanotubes, did not migrate through the gel. We concluded that the pore size was too small for the penetration of large molecules, and that only small molecules could penetrate the gel matrix. The migration mechanism of small molecules was similar to that observed in conventional gel electrophoresis. We concluded that the gel matrix prepared from surfactant is a promising matrix for migration and purification of small molecules. We also expect that the gel can be used as a nanoscale filter to trap large molecules, allowing only small molecules to pass.

  1. Studies of matrix vesicle-induced mineralization in a gelatin gel

    NASA Technical Reports Server (NTRS)

    Boskey, A. L.; Boyan, B. D.; Doty, S. B.; Feliciano, A.; Greer, K.; Weiland, D.; Swain, L. D.; Schwartz, Z.

    1992-01-01

    Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

  2. Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

    NASA Technical Reports Server (NTRS)

    Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor); Janis, Abram D. (Inventor)

    2015-01-01

    The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

  3. Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

    NASA Technical Reports Server (NTRS)

    Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor); Janis, Abram D. (Inventor)

    2014-01-01

    The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

  4. Methods of Manufacturing Bioactive Gels from Extracellular Matrix Material

    NASA Technical Reports Server (NTRS)

    Kentner, Kimberly A. (Inventor); Stuart, Katherine A. (Inventor); Janis, Abram D. (Inventor)

    2016-01-01

    The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

  5. Interaction of electromagnetic fields with chondrocytes in gel culture. Final report, February-August 1989

    SciTech Connect

    Grodzinsky, A.J.; Gluzband, Y.A.; Buschmann, M.D.

    1990-02-01

    The research accomplished during this project period focused on control experiments designed to establish whether cartilage cells from normal cartilage will continue to synthesize and accumulate normal extracellular matrix in agarose gel culture. This information is essential to properly design experiments to qualify changes in chondrocyte biosynthesis due to applied electromagnetic fields. The results suggest that both normal chondrocytes and swarm rat chondrosarcoma cells in agarose culture can continue to synthesize matrix macromolecules at a rate similar to or slightly higher than that in normal cartilage; also, that chondrocytes in agarose can successfully mediate assembly and accumulation of normal, mechanically functional extracellular matrix.

  6. Calcium alginate gel as encapsulation matrix for coimmobilized enzyme systems.

    PubMed

    Blandino, A; Macías, M; Cantero, D

    2003-07-01

    Encapsulation within calcium alginate gel capsules was used to produce a coimmobilized enzyme system. Glucose oxidase (GOD) and catalase (CAT) were chosen as model enzymes. The same values of Vmax and Km app for the GOD encapsulated system and for the GOD-CAT coencapsulated system were calculated. When gel beads and capsules were compared, the same catalyst deactivation sequence for the two enzymes was observed. However, when capsules were employed as immobilization support, GOD efficiencies were higher than for the gel beads. These results were explained in terms of the structure of the capsules.

  7. Thermally reversible gels in electrophoresis. I - Matrix characterization

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Snyder, Robert S.

    1988-01-01

    Two series of thermally reversible hydrogen-bonded gels have been characterized: (5 pct) PVA-(4 pct) PEG and (5 pct) PVA-(0.04 pct) borate gels. They both have extremely low melting points (16-17 C) and could be of potential interest for recovery of proteins after preparative electrophoresis. The PVA-borate gels can be exploited in the pH range 7-11 by progressively increasing the borate content in the pH interval 8 to 7 and concomitantly decreasing the borate levels in the pH zone 8 to 11. It is hypothesized that the low melting point of these gels is due to the fact that they are sparingly and sparsely hydrogen bonded along the PVA chain: on the average, 1 OH group out of 3 or 4 OH groups in the PVA polymer should be engaged in H-bond formation.

  8. Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

    PubMed

    Robinson, Nicholas P

    2013-01-01

    Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

  9. Comparison of oligonucleotide migration in a bicontinuous cubic phase of monoolein and water and in a fibrous agarose hydrogel.

    PubMed

    Sanandaji, Nima; Carlsson, Nils; Voinova, Marina; Akerman, Björn

    2006-08-01

    Porous hydrogels such as agarose are commonly used to analyze DNA and water-soluble proteins by electrophoresis. More recently lyotropic liquid crystals, such as the diamond cubic phase formed by the lipid monoolein and water, has become a new type of well-defined porous structure of interest for both hydrophilic and amphiphilic analytes. Here we compare these two types of matrixes by investigating the nature of retardation they confer to an oligonucleotide that migrates in their respective aqueous phases. The retardation for a 25-mer oligonucleotide was found to be about 35-fold stronger in the cubic phase than in an agarose hydrogel modified to have the same average pore size. According to modelling, the strong retardation is primarily due to the fact that hydrodynamic interaction with the continuous monoolein membrane is a stronger source of friction than the steric interactions (collisions) with discrete gel fibres. A secondary effect is that the regular liquid crystal has a narrower pore-size distribution than the random network of the agarose gel. In agreement with experiments, these two effects together predict that the retardation in the cubic phase is a 30-fold stronger than in an agarose gel with the same average pore radius.

  10. An improved protocol for the preparation and restriction enzyme digestion of pulsed-field gel electrophoresis agarose plugs for the analysis of Legionella isolates.

    PubMed

    Chang, Bin; Amemura-Maekawa, Junko; Watanabe, Haruo

    2009-01-01

    Pulsed-field gel electrophoresis (PFGE), which determines the genomic relatedness of isolates, is currently used for the epidemiological investigation of infectious agents such as bacteria. In particular, this method has been used for the epidemiological investigation of Legionella outbreaks. However, it takes 4 days to complete a Legionella-PFGE analysis. Due to partial digestion and DNA damage, the reproducibility of the obtained fragment digestion patterns is poor for this pathogen. In this study, we report an improved protocol that takes only 2 days to complete and that allows clear discrimination of the restriction profile with higher reproducibility than that previously achieved.

  11. Nitric Oxide Sensors Obtained Through the Entrapment of Iron Complexes in Sol-Gel Matrix

    DTIC Science & Technology

    2002-04-05

    iron complexes in sol-gel matrix Juliana C. Biazzotto, Jodo F. Borin, Roberto Mendonga Faria’ and Carlos F.O Graeff Departamento de Fisica e...Matemdtica-FFCLRP-USP, Av. Bandeirantes 3900, 14040-901 Ribeirdo Preto, Brazil 1-Instituto de Fisica de S~o Carlos-USP, C.P. 369, 13560-970 Sdo Carlos, Brazil

  12. Structural transition in the humic matrix of soil gels and its effect on the soil properties

    NASA Astrophysics Data System (ADS)

    Fedotov, G. N.; Shoba, S. A.; Pozdnyakov, A. I.; Puzanova, A. E.

    2014-09-01

    The analysis of drying-wetting cycles in soils has shown that the existence of the humic matrix of soil gels and, hence, the soil structure is ensured by hydrophilic bonds in dry soils and hydrophobic bonds in wet soils. This suggests that the structural transition from one mechanism controlling the stability of the soil gels and the existence of the soil structure to another mechanism occurs in the humic matrix of soil gels in a specific range of water content. The experimental results have confirmed the effect of the structural transition on the water stability of the soil structure, the pHwater, the hydrophilicity of the soil particle surface, and the structural-mechanical properties of the soils.

  13. Estimating the DNA strand breakage using a fuzzy inference system and agarose gel electrophoresis, a case study with toothed carp Aphanius sophiae exposed to cypermethrin.

    PubMed

    Poorbagher, Hadi; Moghaddam, Maryam Nasrollahpour; Eagderi, Soheil; Farahmand, Hamid

    2016-07-01

    The DNA breakage has been widely used in ecotoxicological studies to investigate effects of pesticides in fishes. The present study used a fuzzy inference system to quantify the breakage of DNA double strand in Aphanius sophiae exposed to the cypermethrin. The specimens were adapted to different temperatures and salinity for 14 days and then exposed to cypermethrin. DNA of each specimens were extracted, electrophoresed and photographed. A fuzzy system with three input variables and 27 rules were defined. The pixel value curve of DNA on each gel lane was obtained using ImageJ. The DNA breakage was quantified using the pixel value curve and fuzzy system. The defuzzified values were analyzed using a three-way analysis of variance. Cypermethrin had significant effects on DNA breakage. Fuzzy inference systems can be used as a tool to quantify the breakage of double strand DNA. DNA double strand of the gill of A. sophiae is sensitive enough to be used to detect cypermethrin in surface waters in concentrations much lower than those reported in previous studies.

  14. Modification of agarose with carboxylation and grafting dopamine for promotion of its cell-adhesiveness.

    PubMed

    Su, Yixue; Chu, Bin; Gao, Yuan; Wu, Chaoxi; Zhang, Lingmin; Chen, Peng; Wang, Xiaoying; Tang, Shunqing

    2013-02-15

    In order to improve bioactivity of agarose, we modified agarose by carboxylation and grafting dopamine. Under alkaline condition, carboxylated agarose was prepared using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation system by oxidizing C(6) hydroxyl on D-galactose ring into carboxyl group, and the maximum value of the degree of carboxylation reached 30%. With the increase of the amount of oxidant, the molecular weight of the carboxylated agarose decreased to 4 kDa by gel permeation chromatography (GPC) measure. Carboxylated agarose reacted with dopamine through EDC condensation reaction to obtain agarose grafting dopamine (Ag-g-DA), and the grafting rate of dopamine was determined to be 9.3% by UV spectroscopy at 280 nm. The structures of these modified agaroses were determined by FT-IR and (13)C NMR. Both carboxylated agarose and Ag-g-DA showed no cytotoxicity and promoted cell-adhesiveness.

  15. In vivo measurement of flavour release from mixed phase gels.

    PubMed

    Taylor, A J; Besnard, S; Puaud, M; Linforth, R S

    2001-05-01

    Flavour release was investigated from pure gelatin, pure agarose and mixed gelatin-agarose gels, all containing 25% sucrose and flavoured with p-cymene, ethyl butyrate, pyrazine and ethanol. Gels were characterised by optical microscopy, and rheological techniques to determine phase separation, elastic modulus and melting temperature. Volatile release was measured by monitoring the four volatiles in the expired air from one individual eating the gels, using Atmospheric Pressure Chemical Ionisation-Mass Spectrometry. The release pattern of p-cymene was not affected by gel type. The release of ethanol, ethyl butyrate and pyrazine was affected to different extents by the matrix suggesting that both the properties of the volatile and the matrix determine volatile release in vivo.

  16. Investigation of optical properties of anthocyanin doped into sol-gel based matrix

    NASA Astrophysics Data System (ADS)

    Hashim, Hasrina; Abdul Aziz, Nik Mohd Azmi Nik; Isnin, Aishah

    2012-06-01

    Anthocyanin dye was extracted from petal of Hibiscus rosasinensis (Bunga Raya) and doped into sol-gel based matrix to investigate an effect of pH change on its optical properties. Sol-gel matrix based on Vinyl triethoxysilene (VTES) as a precursor was prepared through Sol-gel process at pH 7. The sol was doped with 0.1% of Anthocyanin and the same amount of dye was also dissolved in ethanol as a comparative sample. Hydrochloric Acid, HCl and Tetramethylammonium Hydroxide, TMAH were used to change the pH value by adding them at various concentrations into each sample. The emission spectra and chemical structures of the samples were measured by Spectrofluorometer and Fourier Transform Infrared (FTIR) respectively. When excited at 410 nm, two emission peaks at about 492 and 574 nm were observed for Anthocyanin in acidic environment both in ethanol and VTES sol. In base environment however, only Anthocyanin dissolved in ethanol produced emission peak with a single peak at about 539 nm. The sensitivity of Anthocyanin dye toward pH changes in VTES open a possibility to use it as sensing element in which sol-gel based matrix are known to have higher mechanical strength and thermal stability.

  17. Degradation potential of protocatechuate 3,4-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels and on glyoxyl agarose.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Krysiak, Marta; Wojcieszyńska, Danuta

    2014-01-01

    Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5 °C and 10 °C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.

  18. Chitosan/agarose hydrogels: cooperative properties and microfluidic preparation.

    PubMed

    Zamora-Mora, Vanessa; Velasco, Diego; Hernández, Rebeca; Mijangos, Carmen; Kumacheva, Eugenia

    2014-10-13

    The preparation of composite biopolymer hydrogels offers the capability to produce biocompatible and biodegradable materials with cooperative properties. In this paper, two natural polymers, namely, chitosan and agarose were employed to prepare composite hydrogels with dual pH and temperature properties. The elastic modulus of the composite hydrogels increased with agarose concentration reaching the value of 1 kPa for the chitosan/agarose gel with a 2% (w/v) concentration of agarose. In addition, composite gels exhibited a higher stability in acidic aqueous solutions, in comparison with agarose gels. The drug release properties of the composite hydrogels were tested by loading a model anticancer drug, 5-Fluorouracil, in the hydrogel interior. At pH=7.4, the cumulative release of 5-FU was ∼ 50% within 96 h and decreased to ∼ 33% at pH = 5.2, which was attributed to the different solubility of 5-FU as a function of pH. The preparation of composite microgels with controllable dimensions in the range from 42 to 18 μm and with narrow size distribution (polidispersity not exceeding 1.5%) was achieved by the microfluidic emulsification of an aqueous mixture of chitosan and agarose and subsequent gelation of the precursor droplets by cooling.

  19. Immobilization and characterization of the transmembrane ion channel peptide gramicidin in a sol-gel matrix

    NASA Astrophysics Data System (ADS)

    Esquembre, Rocío; Poveda, José Antonio; Mallavia, Ricardo; Mateo, C. Reyes

    2007-05-01

    Immobilization of ion channels requires of a methodology able to retain the physical properties of the lipid bilayer where their activity is performed. However, most of lipid membrane immobilization methods have been observed to alter the structural properties of the bilayers. Use of sol-gel routes seems to be an interesting alternative, although unstable liposomes were obtained when conventional sol-gel methodology was employed for immobilizing. Recently, we have suggested that use of alcohol-free sol-gel routes combined with negatively charged lipids could minimize effects exerted by host matrix on liposome structure, increasing its stability. Here we confirm this assumption by analysing the physical properties of a series of zwitterionic and anionic liposomes entrapped in a sol-gel matrix and we develop a methodology able to retain the physical properties of the lipid bilayer. This methodology has been successfully used to immobilize the transmembrane ion channel peptide gramicidin. Gramicidin was reconstituted in anionic liposomes and its immobilization was confirmed from changes observed in the photophysical properties of the tryptophan residues. Ion channel activity was determined using the fluorescent dye pyrene-1,3,6,8-tetrasulphonic acid (PTSA) and long term stability of the immobilized system was checked from steady-state fluorescence anisotropy measurements.

  20. Supported Molecular Matrix Electrophoresis.

    PubMed

    Matsuno, Yu-Ki; Kameyama, Akihiko

    2015-01-01

    Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique.

  1. Silica sol-gel matrix doped with Photolon molecules for sensing and medical therapy purposes.

    PubMed

    Podbielska, Halina; Ulatowska-Jarza, Agnieszka; Müller, Gerhard; Holowacz, Iwona; Bauer, Joanna; Bindig, Uwe

    2007-11-01

    Photolon is one of the new photosensitisers that has found application in photodynamic therapy (PDT). Its chemical structure has a partially reduced porphyrin moiety and its molecular structure is comparable to chlorin e(6), which can be isolated after hydrolysis of the 5-membered exocyclic beta-ketoester moiety of pheophorbide a. For this study, a Photolon doped sol-gel matrix was produced in the form of coatings deposited on silica fibers cores. The material was produced from sols prepared from the silicate precursor TEOS mixed with ethyl alcohol. The sol-gel films were prepared with factor R=20, where R denotes the solvent-to-precursor molar ratio. Hydrochloric acid was added as a catalyst in the correct proportion to ensure acid hydrolysis (pH approximately 2). The mixture was stirred at room temperature for 4h using a magnetic stirrer (speed 400 rpm). The coated fibers were examined in different environments, liquid and gaseous, at different pH values and with various zinc cation concentrations. The chemical reactions were studied by means of spectroscopic methods, whereby the fluorescence response was studied. It was demonstrated that Photolon immobilized in a sol-gel matrix is accessible for the environment and shows visible response to the external changes. Furthermore, it was observed that these reactions are reversible. These biomaterials are also examined as carriers for PDT. It was also proved that a toxic effect is observed an environment with microorganisms, meaning that doped coatings have photodynamic activity.

  2. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    ERIC Educational Resources Information Center

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels.…

  3. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  4. Glucose microbiosensor based on alumina sol-gel matrix/electropolymerized composite membrane.

    PubMed

    Chen, Xiaohong; Hu, Yibai; Wilson, George S

    2002-12-01

    A procedure is described that provides co-immobilization of enzyme and bovine serum albumin (BSA) within an alumina sol-gel matrix and a polyphenol layer permselective for endogenous electroactive species. BSA has first been employed for the immobilization of glucose oxidase (GOx) on a Pt electrode in a sol-gel to produce a uniform, thin and compact film with enhanced enzyme activity. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)-trimethoxysilane was constructed by electrochemically-assisted crosslinking. This hybrid film outside the enzyme layer contributed both to the improved stability and to permselectivity. The resulting glucose sensor was characterized by a short response time (<10 s), high sensitivity (10.4 nA/mM mm(2)), low interference from endogenous electroactive species, and a working lifetime of at least 60 days.

  5. Extracellular matrix mediates epithelial effects on chondrogenesis in vitro.

    PubMed

    Solursh, M; Jensen, K L; Zanetti, N C; Linsenmayer, T F; Reiter, R S

    1984-10-01

    It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.

  6. Giant unilamellar vesicles formed by hybrid films of agarose and lipids display altered mechanical properties.

    PubMed

    Lira, Rafael B; Dimova, Rumiana; Riske, Karin A

    2014-10-07

    Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.

  7. On-line detection of proteins in gel electrophoresis by ultraviolet absorption and by native fluorescence utilizing a charge-coupled device imaging system

    SciTech Connect

    Koutny, L.B.; Yeung, E.S. )

    1993-01-15

    Slab-gel electrophoresis is the most common technique for the separation of high molecular weight biomolecules such a proteins. Acrylamide gels, as described by Laemmli, are generally the matrix of choice for the separation of SDS-denatured proteins via electrophoresis. Agarose gels, similar to those used for nucleic acids, are also useful for the separation of proteins but have not been widely applied. Agarose gels are advantageous for many reasons including simplicity of gel casting, easy sample recovery, and the fact that it is nontoxic to both the experimenter and the proteins. In the past, agarose was not used because of its poor resolving power at molecular weights below 40,000. New agarose gel systems are available that will resolve proteins ranging from 20,000 to 200,000 with or without SDS denaturing. In this study, agarose gel was chosen for its optical qualities and ability to be cast in an open system that can be imaged as the experiment is running. 17 refs., 7 figs.

  8. Extracellular Matrix Fibronectin Stimulates the Self-Assembly of Microtissues on Native Collagen Gels

    PubMed Central

    Sevilla, Carlos A.; Dalecki, Diane

    2010-01-01

    Fibronectin is an adhesive glycoprotein that is polymerized into extracellular matrices via a tightly regulated, cell-dependent process. Here, we demonstrate that fibronectin matrix polymerization induces the self-assembly of multicellular structures in vitro, termed tissue bodies. Fibronectin-null mouse embryonic fibroblasts adherent to compliant gels of polymerized type I collagen failed to spread or proliferate. In contrast, addition of fibronectin to collagen-adherent fibronectin-null mouse embryonic fibroblasts resulted in a dose-dependent increase in cell number, and induced the formation of three-dimensional (3D) multicellular structures that remained adherent and well-spread on the native collagen substrate. An extensive fibrillar fibronectin matrix formed throughout the microtissue. Blocking fibronectin matrix polymerization inhibited both cell proliferation and microtissue formation, demonstrating the importance of fibronectin fibrillogenesis in triggering cellular self-organization. Cell proliferation, tissue body formation, and tissue body shape were dependent on both fibronectin and collagen concentrations, suggesting that the relative proportion of collagen and fibronectin fibrils polymerized into the extracellular matrix influences the extent of cell proliferation and the final shape of microtissues. These data demonstrate a novel role for cell-mediated fibronectin fibrillogenesis in the formation and vertical assembly of microtissues, and provide a novel approach for engineering complex tissue architecture. PMID:20673131

  9. The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

    NASA Astrophysics Data System (ADS)

    Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

    2016-03-01

    The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

  10. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

    PubMed

    Walenda, Gudrun; Hemeda, Hatim; Schneider, Rebekka K; Merkel, Rudolf; Hoffmann, Bernd; Wagner, Wolfgang

    2012-12-01

    Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

  11. Encapsulation of fluorescence vegetable extracts within a templated sol-gel matrix

    NASA Astrophysics Data System (ADS)

    Lacatusu, Ioana; Badea, Nicoleta; Nita, Rodica; Murariu, Alina; Miculescu, Florin; Iosub, Ion; Meghea, Aurelia

    2010-04-01

    The sol-gel encapsulation of labile substances with specific properties and recognition functions within robust polymer matrices remains a challenging task, despite the considerable research that has been focused on this field. Numerous studies have been reported in the field of sol-gel processes regarding different physical and chemical packing of sensitive biomolecules encapsulated in silica matrix. In this paper the classical sol-gel synthesis has been used under mild conditions in order to minimize denaturizing effects on encapsulated active vegetable extracts from flavones class. The silica templated matrix was obtained by using two types of surfactants with different alkyl chain (didodecyldimethyl-ammonium bromide and trioctadecylmetilammonium bromide) as structure-directing agents for the silicon oxide framework. An organic precursor of silicic acid (triethoxymethylsilane) has been used and it was processed by competitive hydrolysis and polycondensation reactions under controlled directions assured by the presence of oriented template. Silica materials thus obtained are used for encapsulation of two flavonoid samples containing as active principles two sources: rutin and a vegetable extract from Begonia plant. The synthesis of encapsulated nanocompounds has been achieved taking into consideration the specific interaction between the colloidal gel precursors and molecular structures of selected biomolecules. The main objective was to improve the encapsulation conditions for specific biomolecules, searching for the highest stability and functionality without loosing the quality of the flavonoid properties, particularly optical properties like fluorescence. The structural properties of the encapsulated samples have been studied by FT-IR and UV-VIS spectroscopy, thermal analysis and SEM/EDX analysis. The fluorescence experiments showed that, in the case of all four encapsulated samples, the fluorescence spectra manifest a significant increase in intensity

  12. Biopolymer gel matrix as acellular scaffold for enhanced dermal tissue regeneration.

    PubMed

    Judith, Rangasamy; Nithya, Mariappan; Rose, Chellan; Mandal, Asit Baran

    2012-07-01

    Biological grafts have drawbacks such as donor scarcity, disease transmission, tissue infection, while the scaffolds of either collagen or chitosan fabrics fail to become part of the tissue at the wound site, though they favor the formation of connective tissue matrix. This study developed a novel composite consisting of the combination of atelocollagen and chitosan in order to provide a biodegradable molecular matrix in gel form as a biomimetic surface for cell attachment, to promote the wound healing in excision wounds. We found that the topical application of biopolymer composite on the wound promoted cell proliferation, migration and collagen deposition overtime. The enhanced cellular activity in the collagen-chitosan treated wound tissue was also assed by increased levels of Platelet derived growth factor (PDGF) and Nerve growth factor (NGF) associated with elevated levels of antioxidants and decreased level of lipid peroxidation. The acellular matrix-like topical application material is designed to guide the eventual re-establishment of an anatomically normal skin. The results of this study demonstrate the feasibility of multi-cell regeneration on a molecular system that mimics tissue engineering in vivo.

  13. Effects of Matrix Metalloproteinases on the Performance of Platelet Fibrin Gel Spiked With Cardiac Stem Cells in Heart Repair

    PubMed Central

    Shen, Deliang; Tang, Junnan; Hensley, Michael Taylor; Li, Taosheng; Caranasos, Thomas George; Zhang, Tianxia

    2016-01-01

    Stem cells and biomaterials have been studied for therapeutic cardiac repair. Previous studies have shown the beneficial effects of platelet fibrin gel and cardiac stem cells when cotransplanted into rodent hearts with myocardial infarction (MI). We hypothesized that matrix metalloproteinases (MMPs) play an important role in such protection. Thus, the present study is designed to elucidate the effects of MMP inhibition on the therapeutic benefits of intramyocardial injection of platelet fibrin gel spiked with cardiac stem cells (cell-gel) in a rat model of acute MI. In vitro, broad-spectrum MMP inhibitor GM6001 undermines cell spreading and cardiomyocyte contraction. In a syngeneic rat model of myocardial infarction, MMP inhibition blunted the recruitment of endogenous cardiovascular cells into the injected biomaterials, therefore hindering de novo angiogenesis and cardiomyogenesis. Echocardiography and histology 3 weeks after treatment revealed that metalloproteinase inhibition diminished the functional and structural benefits of cell-gel in treating MI. Reduction of host angiogenesis, cardiomyocyte cycling, and MMP-2 activities was evident in animals treated with GM6001. Our findings suggest that MMPs play a critical role in the therapeutic benefits of platelet fibrin gel spiked with cardiac stem cells for treating MI. Significance In this study, the effects of matrix metalloproteinase inhibition on the performance of platelet gel spiked with cardiac stem cells (cell-gel) for heart regeneration are explored. The results demonstrate that matrix metalloproteinases are required for cell-gel to exert its benefits in cardiac repair. Inhibition of matrix metalloproteinases reduces cell engraftment, host angiogenesis, and recruitment of endogenous cardiovascular cells in rats with heart attack. PMID:27112177

  14. Spectrofluorimetric assessment of hydrochlorothiazide using optical sensor nano-composite terbium ion doped in sol-gel matrix.

    PubMed

    Youssef, A O

    2012-05-01

    A new, simple, sensitive and selective spectrofluorimetric method for the determination of Hydrochlorothiazide was developed in acetonitrile at pH 6.2. The Hydrochlorothiazide can remarkably enhance the luminescence intensity of the Tb(3+) ion doped in sol-gel matrix at λ(ex) = 370 nm. The intensity of the emission band of Tb(3+) ion doped in sol-gel matrix was increased due to the energy transfer from the triplet excited state of Hydrochlorothiazide to ((5)D(4)) excited energy state of Tb(3) ion. The enhancement of the emission band of Tb(3+) ion doped in sol-gel matrix at ((5)D(4)→(7)F(5)) 545 nm was directly proportion to the concentration of Hydrochlorothiazide with a dynamic ranges of 5.0 × 10(-10)-5.0 × 10(-6) mol L(-1) and detection limit of 2.2 × 10(-11) mol L(-1).

  15. Removal of digoxin from plasma using monoclonal anti-digoxin antibodies immobilized on agarose

    SciTech Connect

    Brizgys, M.; Pincus, S.; Rollins, D.E.

    1986-05-01

    Monoclonal anti-digoxin antibodies (dig-Ab) have been covalently coupled to agarose supports to evaluate them as part of an extracorporeal device for removal of digoxin from the circulation. The agarose supports studied were Sepharose CL-6B, agarose-polyacrolein microsphere (APAM) beads, Bio Gel A-5m and Affi-gel 15 (Bio-Rad). Antibody concentrations between 2 and 4 mg/g gel were coupled to the agarose beads which were then placed in glass columns. Bovine ..cap alpha..-globulin coupled to the agarose supports was used as a control. Binding capacity and affinity of the immobilized antibody were determined by perfusing the dig-Ab agarose beads with a plasma solution containing /sup 3/H-digoxin and various concentrations of digoxin. The binding capacity of the immobilized dig-Ab was 30% of the theoretical value for Sepharose, Bio Gel and Affigel, and 10% of the theoretical value for dig-Ab coupled to APAM beads. The affinity of the immobilized dig-Ab was 10-100 fold less than non-immobilized Ab (3.4 x 10/sup 8/M/sup -1/. The APAM beads showed a significant decrease in binding of digoxin as the flow rate was increased from 0.5 to 5.0 ml/min. These data demonstrate that dig-Ab coupled to agarose and incorporated into a column can be used to remove digoxin from plasma in vitro.

  16. Zirconium carbonitride pellets by internal sol gel and spark plasma sintering as inert matrix fuel material

    NASA Astrophysics Data System (ADS)

    Hedberg, Marcus; Cologna, Marco; Cambriani, Andrea; Somers, Joseph; Ekberg, Christian

    2016-10-01

    Inert matrix fuel is a fuel type where the fissile material is blended with a solid diluent material. In this work zirconium carbonitride microspheres have been produced by internal sol gel technique, followed by carbothermal reduction. Material nitride purities in the produced materials ranged from Zr(N0.45C0.55) to Zr(N0.74C0.26) as determined by X-ray diffraction and application of Vegard's law. The zirconium carbonitride microspheres have been pelletized by spark plasma sintering (SPS) and by conventional cold pressing and sintering. In all SPS experiments cohesive pellets were formed. Maximum final density reached by SPS at 1700 °C was 87% theoretical density (TD) compared to 53% TD in conventional sintering at 1700 °C. Pore sizes in all the produced pellets were in the μm scale and no density gradients could be observed by computer tomography.

  17. Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

    PubMed

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

  18. Calcium Alginate Gels as Stem Cell Matrix – Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

    PubMed Central

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B.; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs. PMID:25793885

  19. Photoconductivity and stabilization of dopamine embedded in sol-gel TiO2 matrix

    NASA Astrophysics Data System (ADS)

    Prado-Prone, Gina; Valverde-Aguilar, Guadalupe; García-Macedo, Jorge; Vergara-Aragón, P.

    2012-09-01

    This work reports the synthesis of amorphous TiO2 matrix by sol-gel method at atmospheric conditions. DA was encapsulated in a TiO2 matrix to reduce its chemical instability. To TiO2/DA sample was added the 15C5 to diminish the oxidation process. The stabilization process was followed by absorption spectra, colour change and infrared spectroscopy. Oxidation processes of the DA were identified by the presence of DA-quinone and DA-chrome. The TiO2/DA complex retarded the oxidation process for 30 days, while the TiO2/DA/15C5 complex this period was extended for 47 days. Photoconductivity studies were performed on both kinds of samples to analyze their charge transports. The experimental data were fitted with straight lines at darkness and under illumination at 320 nm, 400 nm, and 515 nm. This indicates an ohmic behavior. Transport parameters were calculated. The conductive effect is stronger under darkness than under illumination at 320 nm because the oxidation process in the darkness is less intense than under illumination.

  20. Optimizing the calcium content of a copolymer acrylamide gel matrix for dark-grown seedlings

    NASA Technical Reports Server (NTRS)

    Myers, P. N.; Mitchell, C. A.

    1998-01-01

    A copolymer acrylamide acrylate gel was investigated as the sole root matrix for dark-grown seedlings of soybean (Glycine max Merr. 'Century 84'). Increasing Ca2+ in the hydrating solution of the hydrogel from 1 to 10 mM decreased its water-holding capacity from 97 to 46 mL g-1, yet water potential of the medium remained high, sufficient for normal plant growth at all Ca2+ concentrations tested. Elongation rate of dark-grown soybean seedlings over a 54-hour period was 0.9, 1.5, and 1.8 mm h-1 with 1.0, 2.5, or 5.0 mM Ca2+, respectively, but did not increase with further increases in Ca2+ concentration. Further study revealed that Na+ was released from the hydrogel medium and was taken up by the seedlings as Ca2+ increased in the medium. In dry hypocotyl tissue, sodium content correlated negatively with calcium content. Despite the presence of Na+ in the hydrogel, seedling growth was normal when adequate Ca2+ was added in the hydrating solution. Acrylamide hydrogels hold good potential as a sole growth matrix for short-term experiments with dark-grown seedlings without irrigation.

  1. A biodegradable gel electrolyte for use in high-performance flexible supercapacitors.

    PubMed

    Moon, Won Gyun; Kim, Gil-Pyo; Lee, Minzae; Song, Hyeon Don; Yi, Jongheop

    2015-02-18

    Despite the significant advances in solid polymer electrolytes used for supercapacitors, intractable problems including poor ionic conductivity and low electrochemical performance limit the practical applications. Herein, we report a facile approach to synthesize a NaCl-agarose gel electrolyte for use in flexible supercapacitors. The as-prepared agarose hydrogel consists of a three-dimensional chemically interconnected agarose backbone and oriented interparticular submicropores filled with water. The interconnected agarose matrix acts as a framework that provides mechanical stability to the gel electrolyte and hierarchical porous networks for optimized ion transport. The developed pores with the water filler provide an efficient ionic pathway to the storage sites of electrode. With these properties, the gel electrolyte enables the supercapacitor to have a high specific capacitance of 286.9 F g(-1) and a high rate capability that is 80% of specific capacitance obtained in the case of a liquid electrolyte at 100 mV s(-1). In addition, attributed to the simple procedure and its components, the gel electrolyte is highly scalable, cost-effective, safe, and nontoxic. Thus, the developed gel electrolyte has the potential for use in various energy storage and delivery systems.

  2. Agarose coated spherical micro resonator for humidity measurements.

    PubMed

    Mallik, Arun Kumar; Liu, Dejun; Kavungal, Vishnu; Wu, Qiang; Farrell, Gerald; Semenova, Yuliya

    2016-09-19

    A new type of fiber optic relative humidity (RH) sensor based on an agarose coated silica microsphere resonator is proposed and experimentally demonstrated. Whispering gallery modes (WGMs) in the micro resonator are excited by evanescent coupling using a tapered fiber with ~3.3 µm waist diameter. A change in the relative humidity of the surrounding the resonator air induces changes in the refractive index (RI) and thickness of the Agarose coating layer. These changes in turn lead to a spectral shift of the WGM resonances, which can be related to the RH value after a suitable calibration. Studies of the repeatability, long-term stability, measurement accuracy and temperature dependence of the proposed sensor are carried out. The RH sensitivity of the proposed sensor depends on the concentration of the agarose gel which determines the initial thickness of the deposited coating layer. Studies of the micro- resonators with coating layers fabricated from gels with three different Agarose concentrations of 0.5%, 1.125% and 2.25 wt./vol.% showed that an increase in the initial thickness of the coating material results in an increase in sensitivity but also leads to a decrease of quality factor (Q) of the micro resonator. The highest sensitivity achieved in our experiments was 518 pm/%RH in the RH range from 30% to 70%. The proposed sensor offers the advantages of a very compact form factor, low hysteresis, good repeatability, and low cross sensitivity to temperature.

  3. A simple gel electrophoresis method for separating polyhedral gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Kim, Suhee; Lee, Hye Jin

    2015-07-01

    In this paper, a simple approach to separate differently shaped and sized polyhedral gold nanoparticles (NPs) within colloidal solutions via gel electrophoresis is described. Gel running parameters for separating efficiently gold NPs including gel composition, added surfactant types and applied voltage were investigated. The plasmonic properties and physical structure of the separated NPs extracted from the gel matrix were then investigated using transmission electron microscopy (TEM) and UV-vis spectrophotometry respectively. Data analysis revealed that gel electrophoresis conditions of a 1.5 % agarose gel with 0.1 % sodium dodecyl sulfate (SDS) surfactant under an applied voltage of 100 V resulted in the selective isolation of ~ 50 nm polyhedral shaped gold nanoparticles. Further efforts are underway to apply the method to purify biomolecule-conjugated polyhedral Au NPs that can be readily used for NP-enhanced biosensing platforms.

  4. Dosimetry of {sup 60}Co and {sup 192}Ir gamma-irradiated agarose gels by proton relaxation time measurement and NMR imaging, in a 0-100 Gy dose range

    SciTech Connect

    Chalansonnet, A.; Briguet, A.; Bonnat, J.L.

    1997-05-01

    Localized irradiation of the skin and subcutaneous tissues with large single doses of gamma rays can induce immediate effects characterized by erythema, desquamation, and necrosis. Correlations between the evolution of the lesions and dosimetry studies have to be established by biophysical methods. NMR studies of the effects of an irradiated Fricke solution might be a means of controlling the delivered irradiation doses. After exposition to ionizing radiations, ferrous ions are transformed into ferric ions. Both are paramagnetic ions, and proton spin-lattice relaxation is accelerated depending on the oxidation reaction. In this study, solution of ammonium ferrous sulfate in an acid environment was incorporated into a gelling substance made with agarose, so that T{sub 1} weighted image contrast could be used to detect ferric ion formation. Experiments with {sup 192}Ir and {sup 90}Co gamma rays with doses in the 0 to 100 Gy range were conducted with Fe{sup 2+} concentrations of 0.5, 1, 1.5, and 2 mM in a gelling substance containing 4% agarose. A relationship was established between the amount of Fe{sup 3+} created and the spin-lattice proton relaxation rate, which led to a straightforward dose-effect relation. The use of such high doses allowed us to reproduce realistic conditions of accidental overexposure. A linear relationship was obtained between the doses absorbed and the NMR parameters measured (T{sub 1} and relative image intensity). 17 refs., 3 figs., 1 tab.

  5. Designing Solvent Exchange-Induced In Situ Forming Gel from Aqueous Insoluble Polymers as Matrix Base for Periodontitis Treatment.

    PubMed

    Srichan, Tharatree; Phaechamud, Thawatchai

    2017-01-01

    An in situ forming gel is a dosage form which is promised for site-specific therapy such as periodontal pocket of periodontitis treatment. Ethylcellulose, bleached shellac, and Eudragit RS were applied in this study as a polymeric matrix for in situ forming gel employing N-methyl pyrrolidone (NMP) as solvent. Solutions comprising ethylcellulose, bleached shellac, and Eudragit RS in NMP were evaluated for viscosity, rheology, and rate of water penetration. Ease of administration by injection was determined as the force required to expel polymeric solutions through a needle using texture analyzer. In vitro gel formation and in vitro gel degradation were conducted after injection into phosphate buffer solution pH 6.8. Ethylcellulose, bleached shellac, and Eudragit RS could form the in situ gel, in vitro. Gel viscosity and pH value depended on percentage amount of the polymer, whereas the water diffusion at early period likely relied on types of polymer. Furthermore, the solutions containing higher polymer concentration exhibited the lower degree of degradation. All the preparations were acceptable as injectable dosage forms because the applied force was lower than 50 N. All of them inhibited Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus mutans, and Porphyrommonas gingivalis growth owing to antimicrobial activity of NMP which exhibited a potential use for periodontitis treatment. Moreover, the developed systems presented as the solvent exchange induced in situ forming gel and showed capability to be incorporated with the suitable antimicrobial active compounds for periodontitis treatment which should be further studied.

  6. 3D porous sol-gel matrix incorporated microdevice for effective large volume cell sample pretreatment.

    PubMed

    Lee, Chan Joo; Jung, Jae Hwan; Seo, Tae Seok

    2012-06-05

    In this study, we demonstrated an effective sample pretreatment microdevice that could perform the capture, purification, and release of pathogenic bacteria with a large-volume sample and at a high speed and high-capture yield. We integrated a sol-gel matrix into the microdevice which forms three-dimensional (3D) micropores for the cell solution to pass through and provides a large surface area for the immobilization of antibodies to capture the target Staphylococcus aureus (S. aureus) cells. The antibody was linked to the surface of the sol-gel via a photocleavable linker, allowing the cell-captured antibody moiety to be released by UV irradiation. In addition to the optimization of the antibody immobilization and UV cleavage processes, the cell-capture efficiency was maximized by controlling the sample flow rate with a pumping scheme (3 steps, 5 steps: 3 steps with one flutter step, 7 steps: 3 steps with two flutter steps) and the pumping time (100, 200, and 300 ms). A quantitative capture analysis was performed by targeting a specific gene site of protein A of S. aureus in real-time PCR (RT-PCR). While the 3-step process with an actuation time of 100 ms showed the fastest flow rate (1 mL sample processing time in 10 min), the pumping scheme with the 7-step process and the 300 ms actuation time revealed the highest cell-capture efficiency. A limit of detection study with the 7-step and the 300 ms pumping scheme demonstrated that 100 cells per 100 μL were detected with a 70% yield, and even a single cell could be analyzed via on-chip sample preparation. Thus, our novel sol-gel based microdevice was proven more cost-effective, simple, and efficient in terms of its sample pretreatment ability compared to the use of a conventional 2D flat microdevice. This proposed sample pretreatment device can be further incorporated to an analytical functional unit to realize a micrototal analysis system (μTAS) with sample-in-answer-out capability in the fields of biomedical

  7. Enhancement in dose sensitivity of polymer gel dosimeters composed of radiation-crosslinked gel matrix and less toxic monomers

    NASA Astrophysics Data System (ADS)

    Hiroki, A.; Yamashita, S.; Taguchi, M.

    2015-01-01

    Polymer gel dosimeters based on radiation-crosslinked hydroxypropyl cellulose gel were prepared, which comprised 2-hydroxyethyl methacrylate (HEMA) and polyethylene glycol #400 dimethacrylate (9G) as less toxic monomers and tetrakis (hydroxymethyl) phosphonium chloride (THPC) as an antioxidant. The dosimeters exposed to 60Co γ-rays became cloudy at only 1 Gy. The irradiated dosimeters were optically analyzed by using a UV- vis spectrophotometer to evaluate dose response. Absorbance of the dosimeters linearly increased in the dose range from 0 to 10 Gy, in which dose sensitivity increased with increasing 9G concentration. The dose sensitivity of the dosimeters with 2 wt% HEMA and 3 wt% 9G was also enhanced by increment in THPC.

  8. Concentric gel system to study the biophysical role of matrix microenvironment on 3D cell migration.

    PubMed

    Kurniawan, Nicholas Agung; Chaudhuri, Parthiv Kant; Lim, Chwee Teck

    2015-04-03

    The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell's mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

  9. Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

    PubMed Central

    Kurniawan, Nicholas Agung; Chaudhuri, Parthiv Kant; Lim, Chwee Teck

    2015-01-01

    The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration. PMID:25867104

  10. The effect of an autologous cellular gel-matrix integrated implant system on wound healing

    PubMed Central

    2010-01-01

    Background This manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions. Methods The best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion. Results The preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate (p = 0,06). Conclusion IIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties. PMID:20565787

  11. Monolithic cryogels made of agarose-chitosan composite and loaded with agarose beads for purification of immunoglobulin G.

    PubMed

    Sun, Sijuan; Tang, Yuhai; Fu, Qiang; Liu, Xuan; Guo, Li'an; Zhao, Yanding; Chang, Chun

    2012-05-01

    In order to obtain a novel absorbent with high adsorption capacity for the purification of immunoglobulin G (IgG), continuous supermacroporous agarose beads embedded agarose-chitosan composite monolithic cryogels (agarose-chitosan cryogels) were prepared by cryo-copolymerization of agarose-chitosan blend solutions with glutaraldehyde as the crosslinker in the presence of agarose beads. After coupling 2-mercaptopyridine onto divinylsulfone-activated matrix, the obtained cryogels were used for the purification of IgG. The microstructure morphologies of the cryogels were analyzed by scanning electron microscopy. The results showed that the obtained cryogels possess interconnected pores of 10-100 μm size. The specific surface area was 350 m(2)/g with maximum adsorption capacity of IgG 71.4 mg/g. The cryogels showed workable stability, and can be reused at least 15 times without significant loss in adsorption capacity. IgG purity after one-step purification from human plasma was monitored by electrophoresis and the average recovery was estimated to be 90%.

  12. Matrix molecularly imprinted mesoporous sol-gel sorbent for efficient solid-phase extraction of chloramphenicol from milk.

    PubMed

    Samanidou, Victoria; Kehagia, Maria; Kabir, Abuzar; Furton, Kenneth G

    2016-03-31

    Highly selective and efficient chloramphenicol imprinted sol-gel silica based inorganic polymeric sorbent (sol-gel MIP) was synthesized via matrix imprinting approach for the extraction of chloramphenicol in milk. Chloramphenicol was used as the template molecule, 3-aminopropyltriethoxysilane (3-APTES) and triethoxyphenylsilane (TEPS) as the functional precursors, tetramethyl orthosilicate (TMOS) as the cross-linker, isopropanol as the solvent/porogen, and HCl as the sol-gel catalyst. Non-imprinted sol-gel polymer (sol-gel NIP) was synthesized under identical conditions in absence of template molecules for comparison purpose. Both synthesized materials were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and nitrogen adsorption porosimetry, which unambiguously confirmed their significant structural and morphological differences. The synthesized MIP and NIP materials were evaluated as sorbents for molecularly imprinted solid phase extraction (MISPE) of chloramphenicol in milk. The effect of critical extraction parameters (flow rate, elution solvent, sample and eluent volume, selectivity coefficient, retention capacity) was studied in terms of retention and desorption of chloramphenicol. Competition and cross reactivity tests have proved that sol-gel MIP sorbent possesses significantly higher specific retention and enrichment capacity for chloramphenicol compared to its non-imprinted analogue. The maximum imprinting factor (IF) was found as 9.7, whereas the highest adsorption capacity of chloramphenicol by sol-gel MIP was 23 mg/g. The sol-gel MIP was found to be adequately selective towards chloramphenicol to provide the necessary minimum required performance limit (MRPL) of 0.3 μg/kg set forth by European Commission after analysis by LC-MS even without requiring time consuming solvent evaporation and sample reconstitution step, often considered as an integral part in solid phase extraction work-flow. Intra and

  13. A simple and effective SuperBuffer for DNA agarose electrophoresis.

    PubMed

    Zhang, Jun-He; Wang, Fang; Wang, Tian-Yun

    2011-11-01

    In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses.

  14. Structure and dynamics of spin-labeled insulin entrapped in a silica matrix by the sol-gel method.

    PubMed

    Vanea, E; Gruian, C; Rickert, C; Steinhoff, H-J; Simon, V

    2013-08-12

    The structure and conformational dynamics of insulin entrapped into a silica matrix was monitored during the sol to maturated-gel transition by electron paramagnetic resonance (EPR) spectroscopy. Insulin was successfully spin-labeled with iodoacetamide and the bifunctional nitroxide reagent HO-1944. Room temperature continuous wave (cw) EPR spectra of insulin were recorded to assess the mobility of the attached spin labels. Insulin conformation and its distribution within the silica matrix were studied using double electron-electron resonance (DEER) and low-temperature cw-EPR. A porous oxide matrix seems to form around insulin molecules with pore diameters in the order of a few nanometers. Secondary structure of the encapsulated insulin investigated by Fourier transform infrared spectroscopy proved a high structural integrity of insulin even in the dried silica matrix. The results show that silica encapsulation can be used as a powerful tool to effectively isolate and functionally preserve biomolecules during preparation, storage, and release.

  15. A new preclinical 3-dimensional agarose colony formation assay.

    PubMed

    Kajiwara, Yoshinori; Panchabhai, Sonali; Levin, Victor A

    2008-08-01

    The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

  16. Fabrication of superporous agarose beads for protein adsorption: effect of CaCO3 granules content.

    PubMed

    Du, Kai-Feng; Bai, Shu; Dong, Xiao-Yan; Sun, Yan

    2010-09-10

    Agarose gels were fabricated by water-in-oil emulsification with the addition of CaCO(3) granules at 8-16 wt%. Thus agarose beads of different superporosities were produced after dissolving the solid porogen. The superporous agarose (SA) and homogeneous agarose gels were double cross-linked and modified with diethylaminoethyl chloride to produce anion exchangers. We have proposed to use a superporous replica (porous titania microspheres) to examine the superporous structure and pore size distribution of the soft gel. The replica was prepared with the agarose gel entrapping CaCO(3) granules by a sol-gel-templating method. It was found that the superpores created by CaCO(3) granules were uniformly distributed and ranged from 0.95 microm to 1.33 microm. The physical properties of the gels were significantly affected by the porogen content. Importantly, by increasing the solid porogen to 12 wt%, the bed permeability and effective porosity increased about 48% and 33%, respectively. Further increase in the porogen to 16 wt% led to a decrease of the mechanical strength. With increasing superpores in the beads, the dynamic adsorption capacity of the packed columns increased obviously at 305-916 cm/h. Besides, the column efficiency changed less with increasing flow velocity up to 1200 cm/h. It was concluded that the use of 12 wt% CaCO(3) granules in agarose solution was beneficial for the fabrication of the SA gel with good mechanical stability and promising performance for protein chromatography.

  17. Composites of Quasi-Colloidal Layered Double Hydroxide Nanoparticles and Agarose Hydrogels for Chromate Removal

    PubMed Central

    Gwak, Gyeong-Hyeon; Kim, Min-Kyu; Oh, Jae-Min

    2016-01-01

    Composite hydrogels were prepared that consisted of quasi-colloidal layered double hydroxide (LDH) nanoparticles and agarose via the electrophoretic method, starting from three different agarose concentrations of 0.5, 1, and 2 wt/v%. The composite hydrogel was identified to have a uniform distribution of LDH nanoparticles in agarose matrix. Microscopic studies revealed that the composite hydrogel had a homogeneous quasi-colloidal state of LDHs, while the simple mixture of LDH powder and agarose hydrogels did not. It was determined that agarose concentration of the starting hydrogel did not significantly influence the amount of LDH that developed in the composite. The chromate scavenging efficiency of the composite hydrogel and corresponding agarose or mixture hydrogel was evaluated with respect to time, and chromate concentration. In general, the composite hydrogels exhibited much higher chromate removal efficacy compared with agarose or mixture hydrogels. Through estimating chromate adsorption by LDH moiety in the composite or mixture hydrogel, it was suggested that the agarose component facilitated the stability and dispersibility of the quasi-colloidal state of LDH nanoparticles in the composite resulting in high adsorption efficacy. From Freundlich isotherm adsorption fitting, composites were determined to possess beneficial cooperative adsorption behavior with a high adsorption coefficient. PMID:28344282

  18. Fabrication of high-density collagen fibril matrix gels by renaturation of triple-helix collagen from gelatin.

    PubMed

    Ohyabu, Yoshimi; Yunoki, Shunji; Hatayama, Hirosuke; Teranishi, Yoshikazu

    2013-11-01

    Collagen-based 3-D hydrogels often lack sufficient mechanical strength for tissue engineering. We developed a method for fabrication of high-density collagen fibril matrix (CFM) gels from concentrated solutions of uncleaved gelatin (UCG). Denatured random-coil UCG exhibited more rapid and efficient renaturation into collagen triple-helix than cleaved gelatin (CG) over a broad range of setting temperatures. The UCG solution formed opaque gels with high-density reconstituted collagen fibrils at 28-32 °C and transparent gels similar to CG at <25 °C. The unique gelation properties of UCG enabled the encapsulation of cultured cells in CFM of high solid volume (>5%) and elasticity (1.28 ± 0.15 kPa at 5% and 4.82 ± 0.38 kPa at 8%) with minimal cell loss. The elastic modulus of these gels was higher than that of conventional CFM containing 0.5% collagen. High-strength CFM may provide more durable hydrogels for tissue engineering and regenerative medicine.

  19. A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption.

    PubMed

    Desbois, Linda; Padirac, Adrien; Kaneda, Shohei; Genot, Anthony J; Rondelez, Yannick; Hober, Didier; Collard, Dominique; Fujii, Teruo

    2012-01-01

    Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the "push-pull" method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications.

  20. Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum

    PubMed Central

    2013-01-01

    Background An eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents. Results A large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the

  1. Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels.

    PubMed

    Nicodemus, G D; Skaalure, S C; Bryant, S J

    2011-02-01

    While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression.

  2. Ultra-deep desulfurization via reactive adsorption on peroxophosphomolybdate/agarose hybrids.

    PubMed

    Xu, Jian; Li, Huacheng; Wang, Shengtian; Luo, Fang; Liu, Yunyu; Wang, Xiaohong; Jiang, Zijiang

    2014-09-01

    A catalyst system composed of peroxophosphomolybdates as catalytic center and agarose as matrix material had been designed. The [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]/agarose (C16PMo(O2)2/agarose) hybrid was found to be active for oxidation desulfurization (ODS) of dibenzothiophene (DBT) or real fuel into corresponding sulfone by H2O2 as an oxidant, while the sulfur content could be reduced to 5ppm. The higher activity comes from its components including [PO4{MoO(O2)2}4] catalytic sites, the hydrophobic quaternary ammonium cation affinity to low polarity substrates, and agarose matrix affinity to H2O2 and sulfone. During the oxidative reaction, the mass transfer resistance between H2O2 and organic sulfurs could be decreased and the reaction rate could increase by the assistance of agarose and hydrophobic tails of [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]. Meanwhile, the oxidative products could be adsorbed by agarose matrix to give clean fuel avoiding the post-treatment. In addition, the hybrid was easily regenerated to be reused.

  3. Injectable Amorphous Chitin-Agarose Composite Hydrogels for Biomedical Applications

    PubMed Central

    Priya, Murali Vishnu; Kumar, Rajendran Arun; Sivashanmugam, Amirthalingam; Nair, Shantikumar Vasudevan; Jayakumar, Rangasamy

    2015-01-01

    Injectable hydrogels are gaining popularity as tissue engineering constructs because of their ease of handling and minimal invasive delivery. Making hydrogels from natural polymers helps to overcome biocompatibility issues. Here, we have developed an Amorphous Chitin (ACh)-Agarose (Agr) composite hydrogel using a simpletechnique. Rheological studies, such as viscoelastic behavior (elastic modulus, viscous modulus, yield stress, and consistency), inversion test, and injectability test, were carried out for different ACh-Agr concentrations. The composite gel, having a concentration of 1.5% ACh and 0.25% Agr, showed good elastic modulus (17.3 kPa), yield stress (3.8 kPa), no flow under gravity, injectability, and temperature stability within the physiological range. Based on these studies, the optimum concentration for injectability was found to be 1.5% ACh and 0.25% Agr. This optimized concentration was used for further studies and characterized using FT-IR and SEM. FT-IR studies confirmed the presence of ACh and Agr in the composite gel. SEM results showed that the lyophilized composite gel had good porosity and mesh like networks. The cytocompatibility of the composite gel was studied using human mesenchymal stem cells (hMSCs). The composite gels showed good cell viability.These results indicated that this injectable composite gel can be used for biomedical applications. PMID:26308065

  4. Ultrahigh-throughput approach for analyzing single-cell genomic damage with an agarose-based microfluidic comet array.

    PubMed

    Li, Yiwei; Feng, Xiaojun; Du, Wei; Li, Ying; Liu, Bi-Feng

    2013-04-16

    Genomic DNA damage was generally identified with a "comet assay" but limited by low throughput and poor reproducibility. Here we demonstrated an ultrahigh-throughput approach with a microfluidic chip to simultaneously interrogate DNA damage conditions of up to 10,000 individual cells (approximately 100-fold in throughput over the conventional method) with better reproducibility. For experiment, agarose was chosen as the chip fabrication material, which would further act as an electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. After treatment with different doses of hydrogen peroxide, individual cells were then lysed for subsequent single-cell gel electrophoresis in the direction vertical to microchannel and fluorescence detection. Through morphological analysis and fluorescent measurement of comet-shaped DNA, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method. It indicated that the agarose-based microfluidic comet array electrophoresis was simple, highly reproducible, and of high throughput, providing a new method for highly efficient single-cell genomic analysis.

  5. Cheap glass fiber mats as a matrix of gel polymer electrolytes for lithium ion batteries

    PubMed Central

    Zhu, Yusong; Wang, Faxing; Liu, Lili; Xiao, Shiyin; Yang, Yaqiong; Wu, Yuping

    2013-01-01

    Lithium ion batteries (LIBs) are going to play more important roles in electric vehicles and smart grids. The safety of the current LIBs of large capacity has been remaining a challenge due to the existence of large amounts of organic liquid electrolytes. Gel polymer electrolytes (GPEs) have been tried to replace the organic electrolyte to improve their safety. However, the application of GPEs is handicapped by their poor mechanical strength and high cost. Here, we report an economic gel-type composite membrane with high safety and good mechanical strength based on glass fiber mats, which are separator for lead-acid batteries. The gelled membrane exhibits high ionic conductivity (1.13 mS cm−1), high Li+ ion transference number (0.56) and wide electrochemical window. Its electrochemical performance is evaluated by LiFePO4 cathode with good cycling. The results show this gel-type composite membrane has great attraction to the large-capacity LIBs requiring high safety with low cost. PMID:24216756

  6. Cheap glass fiber mats as a matrix of gel polymer electrolytes for lithium ion batteries

    NASA Astrophysics Data System (ADS)

    Zhu, Yusong; Wang, Faxing; Liu, Lili; Xiao, Shiyin; Yang, Yaqiong; Wu, Yuping

    2013-11-01

    Lithium ion batteries (LIBs) are going to play more important roles in electric vehicles and smart grids. The safety of the current LIBs of large capacity has been remaining a challenge due to the existence of large amounts of organic liquid electrolytes. Gel polymer electrolytes (GPEs) have been tried to replace the organic electrolyte to improve their safety. However, the application of GPEs is handicapped by their poor mechanical strength and high cost. Here, we report an economic gel-type composite membrane with high safety and good mechanical strength based on glass fiber mats, which are separator for lead-acid batteries. The gelled membrane exhibits high ionic conductivity (1.13 mS cm-1), high Li+ ion transference number (0.56) and wide electrochemical window. Its electrochemical performance is evaluated by LiFePO4 cathode with good cycling. The results show this gel-type composite membrane has great attraction to the large-capacity LIBs requiring high safety with low cost.

  7. Quantitation of Alkaline Phosphatase Isoenzymes Using Agarose Containing Wheat Germ Lectin

    DTIC Science & Technology

    1989-07-01

    SIl Quantitation of Alkaline Phosphatase Isoenzymes Using Agarose Containing Wheat Germ Lectin A thesis submitted in partial fulfillment of the...16 Wheat Germ Lectin Electrophoresis to Quantitate Alkaline Phosphatase Isoenzymes ................ 16 Alkaline Phosphatase Isoenzyme...vs Polyacrylamide Gel Electrophoresis ......................... 40 Clinical Correlation Using Wheat Germ Lectin 45 Placental Alkaline Phosphatase

  8. In situ cell-matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials.

    PubMed

    Duncan, Neil A; Bruehlmann, Sabina B; Hunter, Christopher J; Shao, Xinxin; Kelly, Elizabeth J

    2014-01-01

    Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell-matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell-matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (∼2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell-matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.

  9. Argatroban-coupled Affi-Gel matrix for the purification of thrombin from plasma.

    PubMed

    Lefkowitz, Jerry B

    2005-10-01

    Sometimes it is necessary to obtain thrombin from limited amounts of human plasma for laboratory assay. None of the available purification methods easily deals with this subject. The procedure described in the present paper uses a readily available pharmaceutical agent, argatroban, to construct an affinity matrix. Argatroban has a high affinity for thrombin and its thrombin binding is reversible. Prothrombin derived from a Ba(2+) precipitate of human plasma is used as the starting material. The crude prothrombin can be bulk activated to thrombin using taipan-snake (Oxyuranus scutellatus) venom and bound to the argatroban-coupled matrix without further processing steps. The thrombin product eluted from the argatroban matrix is very pure as judged by high specific activity and by electrophoresis. This purification scheme is rapid, yielding purified thrombin within 2 days.

  10. Fabrication of multilayered vascular tissues using microfluidic agarose hydrogel platforms.

    PubMed

    Kinoshita, Keita; Iwase, Masaki; Yamada, Masumi; Yajima, Yuya; Seki, Minoru

    2016-11-01

    Vascular tissues fabricated in vitro are useful tools for studying blood vessel-related cellular physiologies and for constructing relatively large 3D tissues. An efficient strategy for fabricating vascular tissue models with multilayered, branched, and thick structures through the in situ hydrogel formation in fluidic channels is proposed. First, an aqueous solution of RGD-alginate containing smooth muscle cells (SMCs) is introduced into channel structures made of agarose hydrogel, forming a cell-embedding Ca-alginate hydrogel layer with a thickness of several hundred micrometers on the channel surface because of the Ca(2+) ions diffused from the agarose hydrogel matrix. Next, endothelial cells (ECs) are introduced and cultured for up to seven days to form hierarchically organized, multilayered vascular tissues. The factors affecting the thickness of the Ca-alginate hydrogel layer, and prepared several types of microchannels with different morphologies are examined. The fabricated vascular tissue models are easily recovered from the channel by simply detaching the agarose hydrogel plates. In addition, the effect of O2 tension (20 or 80%) on the viability and elastin production of SMCs during the perfusion culture is evaluated. This technique would pave a new way for vascular tissue engineering because it enables the facile production of morphologically in vivo vascular tissue-like structures that can be employed for various biomedical applications.

  11. A green and environment-friendly gel polymer electrolyte with higher performances based on the natural matrix of lignin

    NASA Astrophysics Data System (ADS)

    Gong, Sheng-Dong; Huang, Yun; Cao, Hai-Jun; Lin, Yuan-Hua; Li, Yang; Tang, Shui-Hua; Wang, Ming-Shan; Li, Xing

    2016-03-01

    In order to explore one truly green and environment-friendly gel polymer electrolyte (GPE), the natural biopolymer of lignin is firstly all over the world used as matrix to prepare GPE. The electrolyte membrane based on lignin can be easily fabricated just with lignin, liquid electrolyte and distilled water. Through comprehensive investigation of obtained GPE, it is found that the liquid electrolyte uptake reaches up to 230 wt.%; before 100 °C, GPE does not lose any weight and is thermal stable; at room temperature the ion conductivity is 3.73 mS cm-1; the amazing property of lithium ion transference number is high up to 0.85; GPE expresses complete electrochemical stability before 7.5 V and favorable compatibility with lithium anode; the outstanding cell performance of C-rate and cycle capacity. All these remarkably excellent performances endow lignin with application potential in GPE used in lithium ion batteries (LIBs) with higher performances.

  12. Storage stability of low-fat sodium reduced fresh merguez sausage prepared with olive oil in konjac gel matrix.

    PubMed

    Triki, Mehdi; Herrero, Ana M; Jiménez-Colmenero, Francisco; Ruiz-Capillas, Claudia

    2013-08-01

    This paper evaluates the nutritional values and stability during refrigerated storage of fresh beef merguez sausage as affected by a reformulation process which modified the fat content both by reducing fat (replacing beef fat with konjac gel) and incorporating olive oil (replacing beef fat with olive oil stabilized in a konjac matrix) and by reducing sodium content, replacing sodium chloride with a salt mixture (containing potassium chloride, calcium chloride and magnesium chloride). A preservative (sodium metabisulphite) was also used to extend the shelf-life of the product. The fat was reduced by 32 to 80% and sodium by over 36%. The reformulation did not negatively affect the sensory evaluation. Low microbiota growth rate and biogenic amines were attributed mainly to the presence of sodium metabisulphite. This preservative could be used in the reformulation to enhance safety and/or extend the shelf-life of this type of product.

  13. An antibacterial coating based on a polymer/sol-gel hybrid matrix loaded with silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Rivero, Pedro José; Urrutia, Aitor; Goicoechea, Javier; Zamarreño, Carlos Ruiz; Arregui, Francisco Javier; Matías, Ignacio Raúl

    2011-12-01

    In this work a novel antibacterial surface composed of an organic-inorganic hybrid matrix of tetraorthosilicate and a polyelectrolyte is presented. A precursor solution of tetraethoxysilane (TEOS) and poly(acrylic acid sodium salt) (PAA) was prepared and subsequently thin films were fabricated by the dip-coating technique using glass slides as substrates. This hybrid matrix coating is further loaded with silver nanoparticles using an in situ synthesis route. The morphology and composition of the coatings have been studied using UV-VIS spectroscopy and atomic force microscopy (AFM). Energy dispersive X-ray (EDX) was also used to confirm the presence of the resulting silver nanoparticles within the thin films. Finally the coatings have been tested in bacterial cultures of genus Lactobacillus plantarum to observe their antibacterial properties. It has been experimentally demonstrated that these silver loaded organic-inorganic hybrid films have a very good antimicrobial behavior against this type of bacteria.

  14. A Thermally Conductive Composite with a Silica Gel Matrix and Carbon-Encapsulated Copper Nanoparticles as Filler

    NASA Astrophysics Data System (ADS)

    Lin, Jin; Zhang, Haiyan; Hong, Haoqun; Liu, Hui; Zhang, Xiubin

    2014-07-01

    Core-shell-structured nanocapsules with a copper core encapsulated in a carbon shell (Cu-C) were synthesized by a direct-current arc-discharge method. Morphological and microstructural characterization showed that the Cu-C consisted of a nanosized Cu core and carbon shell, with the carbon shells containing 6 to 15 ordered graphitic layers and amorphous carbon that effectively shield the metallic Cu core from oxidation. A thermally conductive composite was successfully fabricated using a silica gel matrix incorporated with Cu-C filler. The Cu-C nanoparticles were homogeneously dispersed in the silica gel. The effects of Cu-C on the thermal conductivity, electrical resistivity, and coefficient of thermal expansion (CTE) of the composite were investigated. For composites with 6.16 vol.%, 11.04 vol.%, 16.70 vol.%, and 23.34 vol.% Cu-C content, the thermal conductivity at 50°C was 0.32 W/(m K) to 0.77 W/(m K), the electrical resistivity was 1.98 × 109, 3.48 × 107, 302, and 1 Ω m, respectively, while the CTE at 200°C was 3.79 × 10-4 K-1 to 3.44 × 10-4 K-1. The results reveal that the ordered graphitic shells in the Cu-C increased both the thermal and electrical conduction, but decreased the CTE by preventing the Cu cores from expanding.

  15. Chemical tailoring of hybrid sol-gel thick coatings as hosting matrix for functional patterned microstructures.

    PubMed

    Falcaro, Paolo; Costacurta, Stefano; Malfatti, Luca; Buso, Dario; Patelli, Alessandro; Schiavuta, Piero; Piccinini, Massimo; Grenci, Gianluca; Marmiroli, Benedetta; Amenitsch, Heinz; Innocenzi, Plinio

    2011-02-01

    A phenyl-based hybrid organic - inorganic coating has been synthesized and processed by hard X-ray lithography. The overall lithography process is performed in a two-step process only (X-rays exposure and chemical etching). The patterns present high aspect ratio, sharp edges, and high homogeneity. The coating has been doped with a variety of polycyclic aromatic hydrocarbon functional molecules, such as anthracene, pentacene, and fullerene. For the first time, hard X-rays have been combined with thick hybrid functional coatings, using the sol-gel thick film directly as resist. A new technique based on a new material combined with hard X-rays is now available to fabricate optical devices. The effect due to the high-energy photon exposure has been investigated using FT-IR and Raman spectroscopy, laser scanner, optical profilometer, and confocal and electron microscope. High-quality thick hybrid fullerene-doped microstructures have been fabricated.

  16. Analysis of spatial diffusion of ferric ions in PVA-GTA gel dosimeters through magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Marrale, Maurizio; Collura, Giorgio; Gallo, Salvatore; Nici, Stefania; Tranchina, Luigi; Abbate, Boris Federico; Marineo, Sandra; Caracappa, Santo; d'Errico, Francesco

    2017-04-01

    This work focused on the analysis of the temporal diffusion of ferric ions through PVA-GTA gel dosimeters. PVA-GTA gel samples, partly exposed with 6 MV X-rays in order to create an initial steep gradient, were mapped using magnetic resonance imaging on a 7T MRI scanner for small animals. Multiple images of the gels were acquired over several hours after irradiation and were analyzed to quantitatively extract the signal profile. The spatial resolution achieved is 200 μm and this makes this technique particularly suitable for the analysis of steep gradients of ferric ion concentration. The results obtained with PVA-GTA gels were compared with those achieved with agarose gels, which is a standard dosimetric gel formulation. The analysis showed that the diffusion process is much slower (more than five times) for PVA-GTA gels than for agarose ones. Furthermore, it is noteworthy that the diffusion coefficient value obtained through MRI analysis is significantly consistent with that obtained in separate study Marini et al. (Submitted for publication) using a totally independent method such as spectrophotometry. This is a valuable result highlighting that the good dosimetric features of this gel matrix not only can be reproduced but also can be measured through independent experimental techniques based on different physical principles.

  17. Mediator-free phenol sensor based on titania sol-gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method.

    PubMed

    Yu, Jiuhong; Liu, Songqin; Ju, Huangxian

    2003-12-30

    A novel amperometric phenol sensor was constructed by immobilizing tyrosinase in a titania sol-gel matrix. The tyrosinase entrapped sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process and avoided the shrinkage and cracking of conventional sol-gel-derived glasses. This matrix provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenol could be oxidized by dissolving oxygen in presence of immobilized tyrosinase to form a detectable product, which was determined at -150 mV without any mediator. The phenol sensor exhibited a fast response (less than 5 s) and sensitivity as high as 103 microA/mM, which resulted from the porous structure and high enzyme loading of the sol-gel matrix. The linear range for phenol determination was from 1.2x10(-7) to 2.6x10(-4) M with a detection limit of 1.0x10(-7) M. The apparent Michaelis-Menten constant of the encapsulated tyrosinase was calculated to be (0.29+/-0.02) mM. The stability of the biosensor was also evaluated.

  18. Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose

    SciTech Connect

    Armstrong, G.D.; Peppler, M.S.

    1987-05-01

    We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.

  19. Three-Dimensional Nanoporous Cellulose Gels as a Flexible Reinforcement Matrix for Polymer Nanocomposites.

    PubMed

    Shi, Zhuqun; Huang, Junchao; Liu, Chuanjun; Ding, Beibei; Kuga, Shigenori; Cai, Jie; Zhang, Lina

    2015-10-21

    With the world's focus on utilization of sustainable natural resources, the conversion of wood and plant fibers into cellulose nanowhiskers/nanofibers is essential for application of cellulose in polymer nanocomposites. Here, we present a novel fabrication method of polymer nanocomposites by in-situ polymerization of monomers in three-dimensionally nanoporous cellulose gels (NCG) prepared from aqueous alkali hydroxide/urea solution. The NCG have interconnected nanofibrillar cellulose network structure, resulting in high mechanical strength and size stability. Polymerization of the monomer gave P(MMA/BMA)/NCG, P(MMA/BA)/NCG nanocomposites with a volume fraction of NCG ranging from 15% to 78%. SEM, TEM, and XRD analyses show that the NCG are finely distributed and preserved well in the nanocomposites after polymerization. DMA analysis demonstrates a significant improvement in tensile storage modulus E' above the glass transition temperature; for instance, at 95 °C, E' is increased by over 4 orders of magnitude from 0.03 MPa of the P(MMA/BMA) up to 350 MPa of nanocomposites containing 15% v/v NCG. This reinforcement effect can be explained by the percolation model. The nanocomposites also show remarkable improvement in solvent resistance (swelling ratio of 1.3-2.2 in chloroform, acetone, and toluene), thermal stability (do not melt or decompose up to 300 °C), and low coefficients of thermal expansion (in-plane CTE of 15 ppm·K(-1)). These nanocomposites will have great promising applications in flexible display, packing, biomedical implants, and many others.

  20. Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

    PubMed

    Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

    2012-04-17

    A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

  1. Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution.

    PubMed

    Allenspach, A L; Kraemer, T G

    1989-04-01

    Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Agarose-based microfluidic device for point-of-care concentration and detection of pathogen.

    PubMed

    Li, Yiwei; Yan, Xinghua; Feng, Xiaojun; Wang, Jie; Du, Wei; Wang, Yachao; Chen, Peng; Xiong, Liang; Liu, Bi-Feng

    2014-11-04

    Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 μL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 μL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.

  3. Enhancement of in vitro and in vivo function of agarose-encapsulated porcine islets by changes in the islet microenvironment.

    PubMed

    Holdcraft, Robert W; Gazda, Lawrence S; Circle, Lisa; Adkins, Hollie; Harbeck, Steven G; Meyer, Eric D; Bautista, Melissa A; Martis, Prithy C; Laramore, Melissa A; Vinerean, Horatiu V; Hall, Richard D; Smith, Barry H

    2014-01-01

    The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose-agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40-50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including β-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

  4. Electrode films of porous agarose: The effects of physical structure on electron transport processes. [Impregnated with Nafion; immobilized electroactive species

    SciTech Connect

    Moran, K.D.

    1988-02-01

    Potential use of chemically modified electrodes in electrocatalysis has stimulated interest in creation and characterization of electrode films for reagent immobilization. We have created two highly porous electrocatalyst support matrices, with high rates of electron transport. Both are based on immobilization of reagents in agarose gel. In one case, Nafion was impregnated into agarose gel films. Diffusion of methyl viologen in Nafionagarose matrices are higher than in Nafion. In Nafion, the diffusion coefficient decreases with increasing methyl viologen concentration, while in Nafionagarose, the opposite dependence is observed. The faster rate of electron transport in Nafionagarose films is related to the heterogeneous structure and the coupling of the diffusion pathways. In the second application of agarose gels as an electrode coating material, agarose hydroxyl groups were activated in 1,1'carbonyldiimidazole and subsequently reacted with amine derivatives of electroactive mediators. Electron transport between the electroactive sites in the gel is very rapid (on the order of 10/sup -7/ cm/sup 2/s. Interpreting the data in light of the Dahms-Ruff description of electron transport shows that the rate of electron transport through both ferrocene and viologen derivatized gels is limited by the rate of electron self-exchange of the species. 22 figs., 15 tabs

  5. Online matrix removal platform for coupling gel-based separations to whole protein electrospray ionization mass spectrometry.

    PubMed

    Kim, Ki Hun; Compton, Philip D; Tran, John C; Kelleher, Neil L

    2015-05-01

    A fractionation method called gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) has been used to dramatically increase the number of proteins identified in top-down proteomic workflows; however, the technique involves the use of sodium dodecyl sulfate (SDS), a surfactant that interferes with electrospray ionization. Therefore, an efficient removal of SDS is absolutely required prior to mass analysis. Traditionally, methanol/chloroform precipitation and spin columns have been used, but they lack reproducibility and are difficult to automate. Therefore, we developed an in-line matrix removal platform to enable the direct analysis of samples containing SDS and salts. Only small molecules like SDS permeate a porous membrane and are removed in a manner similar to cross-flow filtration. With this device, near-complete removal of SDS is accomplished within 5 min and proteins are subsequently mobilized into a mass spectrometer. The new platform was optimized for the analysis of GELFrEE fractions enriched for histones extracted from human HeLa cells. All four core histones and their proteoforms were detected in a single spectrum by high-resolution mass spectrometry. The new method versus protein precipitation/resuspension showed 2- to 10-fold improved signal intensities, offering a clear path forward to improve proteome coverage and the efficiency of top-down proteomics.

  6. Fenugreek hydrogel-agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection.

    PubMed

    Kestwal, Rakesh Mohan; Bagal-Kestwal, Dipali; Chiang, Been-Huang

    2015-07-30

    A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel-agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10-20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel-agarose-acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples.

  7. Mullins effect behaviour under compression in micelle-templated silica and micelle-templated silica/agarose systems.

    PubMed

    Puértolas, J A; Vadillo, J L; Sánchez-Salcedo, S; Nieto, A; Gómez-Barrena, E; Vallet-Regí, M

    2012-02-01

    The mechanical properties of bioceramic conformed pieces based on micelle-templated silica (MTS) such as SBA15, MCM41 and MCM48 as well as MTS/agarose systems have been evaluated under static and cyclic compressive tests. The MTS pieces exhibited a brittle behaviour. Agarose, a biocompatible and biodegradable hydrogel, has been used to shape ceramic-agarose pieces following a low temperature shaping method. Agarose conferred toughness, ductility and a rubbery consistency up to a 60% strain in ceramic MTS/agarose systems leading to a maximum strength of 10-50 MPa, without losing their initial cylindrical structure. This combination of ceramic and organic matrix contributes to avoiding the inherent brittleness of the bioceramic and enhances the compression resistance of hydrogel. The presence of mechanical hysteresis, permanent deformation after the first cycle and recovery of the master monotonous curve of MTS/agarose systems indicate a Mullins-like effect similar to that found in carbon-filled rubber systems. We report this type of mechanical behaviour, the Mullins effect, for the first time in MTS bioceramics and MTS bioceramic/agarose systems.

  8. A combined approach of enamel matrix derivative gel and autogenous bone grafts in treatment of intrabony periodontal defects. A case report.

    PubMed

    Leung, George; Jin, Lijian

    2003-04-01

    Enamel matrix derivative (EMD) has recently been introduced as a new modality in regenerative periodontal therapy. This case report demonstrates a combined approach in topical application of EMD gel (Emdogain) and autogenous bone grafts for treatment of intrabony defects and furcation involvement defects in a patient with chronic periodontitis. The seven-month post-surgery clinical and radiographic results were presented. The combined application of EMD gel with autogenous bone grafts in intrabony osseous defects resulted in clinically significant gain of attachment on diseased root surfaces and bone fill on radiographs. Further controlled clinical studies are required to confirm the long-term effectiveness of the combination of EMD gel and autogenous bone grafts in treatment of various osseous defects in subjects with chronic periodontitis.

  9. Functional modification of agarose: a facile synthesis of a fluorescent agarose-tryptophan based hydrogel.

    PubMed

    Kondaveeti, Stalin; Prasad, Kamalesh; Siddhanta, A K

    2013-08-14

    Microwave assisted facile synthesis of a fluorescent agarose-l-tryptophan hydrogel material employing carbodiimide chemistry (dicyclohexylcarbodiimide/4-dimethylaminopyridine; DCC/DMAP) has been described. The product formed fluorescent hydrogel at 1-1.5% (w/v), exhibiting fluorescence emission in water (λmax 350 nm; 1x10(-4)M), which was significantly higher (ca. 65%) than that of tryptophan at the same concentration. Subsequently, the agarose ester was cross linked with the natural cross linker genipin to yield a blue hydrogel (G-Ag-TrpEst), confirming thereby the insertion of tryptophan moiety on to agarose backbone. Both the ester and cross linked hydrogels demonstrated gelling characteristics similar to agarose and were stable across a wide range of pH media (pHs 1.2, 7.0 and 12.5) under ambient conditions. These tryptophan containing fluorescent hydrogel materials may find applications in biomedical and pharmaceutical industries as potential radical scavengers and sensors.

  10. Immobilization and characterization of 2,3-diaminonaphthalene/cyclodextrin complexes in a sol-gel matrix: a new fluorimetric sensor for nitrite.

    PubMed

    Martínez-Tomé, M J; Esquembre, R; Mallavia, R; Mateo, C R

    2009-01-01

    The aromatic diamino compound 2,3-diaminonaphthalene (DAN) has been extensively used to detect and quantify nitrite ions in biological and environmental samples. We have immobilized the DAN reagent in a porous silicate glass matrix, via previous incorporation of the dye in HP-beta-CD. Changes in fluorescence intensity were used to characterize the inclusion complexes and determine the association constant and stoichiometry of the process. Fluorescence spectrum of these complexes was also used to monitor their immobilization within the sol-gel matrix. Reactivity of the immobilized complexes was evaluated with increasing concentrations of nitrite up to 10 microM (with a detection limit around 20 nM). Results show that sol-gel immobilization does not modify the reactivity of the dye against nitrite and serves to prepare a highly sensitive ready to use fluorescence-based sensor for the specific measurement of nitrite at submicromolar concentrations with no further sample pretreatment.

  11. [Kinetic study on the in situ synthesis of nickle phthalocyanine in silica gel glass matrix by UV/Vis absorption spectra].

    PubMed

    Huang, Juan; Zheng, Chan; Feng, Miao; Zhan, Hong-Bing

    2009-01-01

    In decades, metallo-phthalocyanines (MPcs) have undergone a renaissance because of their singular and unconventional physical properties. However, for the successful application of MPcs in practical devices, it is important to disperse MPc molecules into solid state matrix to fabricate MPc doped composite with desired properties. Inorganic glass is an ideal matrix because of its transparency and high environmental stability. One attractive approach to fabricating MPc/inorganic composite is sol-gel technique. In the present paper, silica gel glass matrix was prepared by hydrolysis and poly-condensation of tetraethyloxysilane. 1,2-dicyanobenzene and analytically pure soluble nickle salt were used as the nickle phthalocyanine (NiPc) reactants and chemical synthesis technique was used to prepare NiPc doped sol-gel materials at several temperatures. During the heat treatment, four 1, 2-dicyanobenzene molecules and one nickle ion collide to form a NiPc molecule. In-situ synthesizing process of NiPc in the pores of silica gel glass matrix was traced by UV/Vis absorption spectra. Owing to the remarkable absorption band of NiPc in visible region, quantity of in-situ synthesized NiPc was calculated by the absorbance at certain wavelength of 670 nm, using composites with physically doped NiPc as a reference. The in-situ synthesized kinetics was studied in detail and found to be consistent with Avrami-Erofeev equation The reaction grades were deduced to be 4.5, 4.5, 3.7, 3.2 and 1.9 respectively at temperatures of 180 degrees C, 185 degrees C, 190 degrees C, 195 degrees C and 200 degrees C, respectively.

  12. A functional agarose-hydroxyapatite scaffold for osteochondral interface regeneration

    PubMed Central

    Khanarian, Nora T.; Haney, Nora M.; Burga, Rachel A.; Lu, Helen H.

    2013-01-01

    Regeneration of the osteochondral interface is critical for integrative and functional cartilage repair. This study focuses on the design and optimization of a hydrogel-ceramic composite scaffold of agarose and hydroxyapatite (HA) for calcified cartilage formation. The first study objective was to compare the effects of HA on non-hypertrophic and hypertrophic chondrocytes cultured in the composite scaffold. Specifically, cell growth, biosynthesis, hypertrophy, and scaffold mechanical properties were evaluated. Next, the ceramic phase of the scaffold was optimized in terms of particle size (200 nm vs. 25 µm) and dose (0–6 w/v%). It was observed that while deep zone chondrocyte (DZC) biosynthesis and hypertrophy remained unaffected, hypertrophic chondrocytes measured higher matrix deposition and mineralization potential with the addition of HA. Most importantly, higher matrix content translated into significant increases in both compressive and shear mechanical properties. While cell hypertrophy was independent of ceramic size, matrix deposition was higher only with the addition of micron-sized ceramic particles. In addition, the highest matrix content, mechanical properties and mineralization potential were found in scaffolds with 3% micro-HA, which approximates both the mineral aggregate size and content of the native interface. These results demonstrate that the biomimetic hydrogel-ceramic composite is optimal for calcified cartilage formation and is a promising design strategy for osteochondral interface regeneration. PMID:22531222

  13. Crosslinking of agarose bioplastic using citric acid.

    PubMed

    Awadhiya, Ankur; Kumar, David; Verma, Vivek

    2016-10-20

    We report chemical crosslinking of agarose bioplastic using citric acid. Crosslinking was confirmed using Fourier transform infrared (FTIR) spectroscopy. The effects of crosslinking on the tensile strength, swelling, thermal stability, and degradability of the bioplastic were studied in detail. The tensile strength of the bioplastic films increased from 25.1MPa for control films up to a maximum of 52.7MPa for citric acid crosslinked films. At 37°C, the amount of water absorbed by crosslinked agarose bioplastic was only 11.5% of the amount absorbed by non-crosslinked controls. Thermogravimetric results showed that the crosslinked samples retain greater mass at high temperature (>450°C) than control samples. Moreover, while the crosslinked films were completely degradable, the rate of degradation was lower compared to non-crosslinked controls.

  14. Rheological Characterization of Ethanolamine Gel Propellants

    NASA Astrophysics Data System (ADS)

    V. S Jyoti, Botchu; Baek, Seung Wook

    2016-07-01

    Ethanolamine is considered to be an environmentally friendly propellant system because it has low toxicity and is noncarcinogenic in nature. In this article, efforts are made to formulate and prepare ethanolamine gel systems, using pure agarose and hybrids of paired gelling agents (agarose + polyvinylpyrrolidine (PVP), agarose + SiO2, and PVP + SiO2), that exhibit a measurable yield stress, thixotropic behavior under shear rate ranges of 1-1,000 s-1 and a viscoelastic nature. To achieve these goals, multiple rheological experiments (including flow and dynamic studies) are performed. In this article, results are presented from experiments measuring the apparent viscosity, yield stress, thixotropy, dynamic strain, frequency sweep, and tan δ behaviors, as well as the effects of the test temperature, in the gel systems. The results show that the formulated ethanolamine gels are thixotropic in nature with yield stress between 30 and 60 Pa. The apparent viscosity of the gel decreases as the test temperature increases, and the apparent activation energy is the lowest for the ethanolamine-(PVP + SiO2) gel system. The dynamic rheology study shows that the type of gellant, choice of hybrid gelling materials and their concentration, applied frequencies, and strain all vitally affect the viscoelastic properties of the ethanolamine gel systems. In the frequency sweep experiment, the ethanolamine gels to which agarose, agarose + PVP, and agarose + SiO2 were added behave like linear frequency-dependent viscoelastic liquids, whereas the ethanolamine gel to which PVP + SiO2 was added behaves like a nearly frequency-independent viscoelastic solid. The variation in the tan δ of these gelled propellants as a function of frequency is also discussed.

  15. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    NASA Astrophysics Data System (ADS)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels. When forced through a maze of holes, the molecule unravels, forming a long chain, slithering through the field of pores in a process colloquially coined "reputation." As a result, the smaller molecules travel farther through the gel when compared to molecules of larger molecular weight. This highly effective "molecular sieve" provides consistent data and allows scientists to compare similar sequences of DNA base pairs in a routine fashion.2 When performed at the high school level, gel electrophoresis provides students the opportunity to learn about a contemporary lab technique of great scientific relevance. Doing real science certainly excites students and motivates them to learn more.

  16. Layer-by-Layer Self-Assembling Gold Nanorods and Glucose Oxidase onto Carbon Nanotubes Functionalized Sol-Gel Matrix for an Amperometric Glucose Biosensor

    PubMed Central

    Wu, Baoyan; Hou, Shihua; Miao, Zhiying; Zhang, Cong; Ji, Yanhong

    2015-01-01

    A novel amperometric glucose biosensor was fabricated by layer-by-layer self-assembly of gold nanorods (AuNRs) and glucose oxidase (GOD) onto single-walled carbon nanotubes (SWCNTs)-functionalized three-dimensional sol-gel matrix. A thiolated aqueous silica sol containing SWCNTs was first assembled on the surface of a cleaned Au electrode, and then the alternate self-assembly of AuNRs and GOD were repeated to assemble multilayer films of AuNRs-GOD onto SWCNTs-functionalized silica gel for optimizing the biosensor. Among the resulting glucose biosensors, the four layers of AuNRs-GOD-modified electrode showed the best performance. The sol-SWCNTs-(AuNRs-GOD)4/Au biosensor exhibited a good linear range of 0.01–8 mM glucose, high sensitivity of 1.08 μA/mM, and fast amperometric response within 4 s. The good performance of the proposed glucose biosensor could be mainly attributed to the advantages of the three-dimensional sol-gel matrix and stereo self-assembly films, and the natural features of one-dimensional nanostructure SWCNTs and AuNRs. This study may provide a new facile way to fabricate the enzyme-based biosensor with high performance.

  17. Layer-by-Layer Self-Assembling Gold Nanorods and Glucose Oxidase onto Carbon Nanotubes Functionalized Sol-Gel Matrix for an Amperometric Glucose Biosensor.

    PubMed

    Wu, Baoyan; Hou, Shihua; Miao, Zhiying; Zhang, Cong; Ji, Yanhong

    2015-09-18

    A novel amperometric glucose biosensor was fabricated by layer-by-layer self-assembly of gold nanorods (AuNRs) and glucose oxidase (GOD) onto single-walled carbon nanotubes (SWCNTs)-functionalized three-dimensional sol-gel matrix. A thiolated aqueous silica sol containing SWCNTs was first assembled on the surface of a cleaned Au electrode, and then the alternate self-assembly of AuNRs and GOD were repeated to assemble multilayer films of AuNRs-GOD onto SWCNTs-functionalized silica gel for optimizing the biosensor. Among the resulting glucose biosensors, the four layers of AuNRs-GOD-modified electrode showed the best performance. The sol-SWCNTs-(AuNRs- GOD)₄/Au biosensor exhibited a good linear range of 0.01-8 mM glucose, high sensitivity of 1.08 μA/mM, and fast amperometric response within 4 s. The good performance of the proposed glucose biosensor could be mainly attributed to the advantages of the three-dimensional sol-gel matrix and stereo self-assembly films, and the natural features of one-dimensional nanostructure SWCNTs and AuNRs. This study may provide a new facile way to fabricate the enzyme-based biosensor with high performance.

  18. Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering.

    PubMed

    Rennerfeldt, Deena A; Renth, Amanda N; Talata, Zsolt; Gehrke, Stevin H; Detamore, Michael S

    2013-11-01

    Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells.

  19. Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering

    PubMed Central

    Rennerfeldt, DA; Renth, AN; Talata, Z; Gehrke, SH; Detamore, MS

    2013-01-01

    Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells. PMID:23932504

  20. Highly efficient up-conversion and bright white light in RE co-doped KYF4 nanocrystals in sol-gel silica matrix

    NASA Astrophysics Data System (ADS)

    Méndez-Ramos, J.; Yanes, A. C.; Santana-Alonso, A.; del-Castillo, J.

    2013-01-01

    Transparent nano-glass-ceramics comprising Yb3+, Er3+ and Tm3+ co-doped KYF4 nanocrystals have been developed from sol-gel method. A structural analysis by means of X-ray diffraction confirmed the precipitation of cubic KYF4 nanocrystals into a silica matrix. Visible luminescence has been analyzed as function of treatment temperature of precursor sol-gel glasses. Highly efficient up-conversion emissions have been obtained under 980 nm excitation and studied by varying the doping level, processing temperature and pump power. Color tuneability has been quantified in terms of CIE diagram and in particular, a white-balanced overall emission has been achieved for a certain doping level and thermal treatment.

  1. Agarose hydrogels embedded with pH-responsive diblock copolymer micelles for triggered release of substances.

    PubMed

    Jin, Naixiong; Morin, Emily A; Henn, Daniel M; Cao, Yu; Woodcock, Jeremiah W; Tang, Shuangcheng; He, Wei; Zhao, Bin

    2013-08-12

    Hybrid agarose hydrogels embedded with pH-responsive diblock copolymers micelles were developed to achieve functional hydrogels capable of stimulus-triggered drug release. Specifically, a well-defined poly(ethylene oxide) (PEO)-based diblock copolymer, PEO-b-poly(2-(N,N-diisopropylamino)ethyl methacrylate) (PEO(113)-b-PDPAEMA(31), where the subscripts represent the degrees of polymerization of two blocks), was synthesized by atom transfer radical polymerization. PDPAEMA is a pH-responsive polymer with a pKa value of 6.3. The PEO(113)-b-PDPAEMA(31) micelles were formed by a solvent-switching method, and their pH-dependent dissociation behavior was investigated by dynamic light scattering and fluorescence spectroscopy. Both studies indicated that the micelles were completely disassembled at pH = 6.40. The biocompatibility of PEO(113)-b-PDPAEMA(31) micelles was demonstrated by in vitro primary cortical neural culture. Hybrid agarose hydrogels were made by cooling 1.0 wt % agarose solutions that contained various amounts of PEO(113)-b-PDPAEMA(31) micelles at either 2 or 4 °C. Rheological measurements showed that the mechanical properties of gels were not significantly adversely affected by the incorporation of diblock copolymer micelles with a concentration as high as 5.0 mg/g. Using Nile Red as a model hydrophobic drug, its incorporation into the core of diblock copolymer micelles was demonstrated. Characterized by fluorescent spectroscopy, the release of Nile Red from the hybrid hydrogel was shown to be controllable by pH due to the responsiveness of the block copolymer micelles. Based on the prominent use of agarose gels as scaffolds for cell transplantation for neural repair, the hybrid hydrogels embedded with stimuli-responsive block copolymer micelles could allow the controlled delivery of hydrophobic neuroprotective agents to improve survival of transplanted cells in tune with signals from the surrounding pathological environment.

  2. One-step preparation of CdS nanocrystals supported on thiolated silica-gel matrix and evaluation of photocatalytic performance.

    PubMed

    Andrade, George R S; Nascimento, Cristiane C; Neves, Erick C; Barbosa, Cintya D'Angeles Espirito Santo; Costa, Luiz P; Barreto, Ledjane S; Gimenez, Iara F

    2012-02-15

    Here we report the use of a thiol-functionalized silica-gel to prepare supported CdS nanocrystals by a facile one-step procedure. Upon changing the relative proportion of the matrix we obtained nanocrystals with different average sizes and size distributions, as evidenced by spectroscopic measurements as well as TEM images. Photoluminescence spectra also indicated that the main effect of the matrix is related to the size control since the spectral profiles were found to be strongly dependent on the excitation wavelength. The performance of the material in the photocatalytic degradation of two commercial dyes (methylene blue and rhodamine 6G) has been tested under sunlight radiation, showing promising results. Almost complete decolorization has been observed after 80 min of exposure, with no adsorption on the silica surface.

  3. Immobilization of Lipases on Heterofunctional Octyl-Glyoxyl Agarose Supports: Improved Stability and Prevention of the Enzyme Desorption.

    PubMed

    Rueda, N; Dos Santos, J C S; Torres, R; Ortiz, C; Barbosa, O; Fernandez-Lafuente, R

    2016-01-01

    Lipases are among the most widely used enzymes in industry. Here, a novel method is described to rationally design the support matrix to retain the enzyme on the support matrix without leaching and also activate the enzyme for full activity retention. Lipases are interesting biocatalysts because they show the so-called interfacial activation, a mechanism of action that has been used to immobilize lipases on hydrophobic supports such as octyl-agarose. Thus, adsorption of lipases on hydrophobic surfaces is very useful for one step purification, immobilization, hyperactivation, and stabilization of most lipases. However, lipase molecules may be released from the support under certain conditions (high temperature, organic solvents), as there are no covalent links between the enzyme and the support matrix. A heterofunctional support has been proposed in this study to overcome this problem, such as the heterofunctional glyoxyl-octyl agarose beads. It couples the numerous advantages of the octyl-agarose support to covalent immobilization and creates the possibility of using the biocatalyst under any experimental conditions without risk of enzyme desorption and leaching. This modified support may be easily prepared from the commercially available octyl-agarose. Preparation of this useful support and enzyme immobilization on it via covalent linking is described here. The conditions are described to increase the possibility of achieving at least one covalent attachment between each enzyme molecule and the support matrix.

  4. In Situ Observations of Thermoreversible Gelation and Phase Separation of Agarose and Methylcellulose Solutions under High Pressure.

    PubMed

    Kometani, Noritsugu; Tanabe, Masahiro; Su, Lei; Yang, Kun; Nishinari, Katsuyoshi

    2015-06-04

    Thermoreversible sol-gel transitions of agarose and methylcellulose (MC) aqueous solutions on isobaric cooling or heating under high pressure up to 400 MPa have been investigated by in situ observations of optical transmittance and falling-ball experiments. For agarose, which undergoes the gelation on cooling, the application of pressure caused a gradual rise in the cloud-point temperature over the whole pressure range examined, which is almost consistent with the pressure dependence of gelling temperature estimated by falling-ball experiments, suggesting that agarose gel is stabilized by compression and that the gelation occurs nearly in parallel with phase separation under ambient and high-pressure conditions. For MC, which undergoes the gelation on heating, the cloud-point temperature showed a slight rise with an initial elevation of pressure up to ∼150 MPa, whereas it showed a marked depression above 200 MPa. In contrast, the gelling temperature of MC, which is nearly identical to the cloud-point temperature at ambient pressure, showed a monotonous rise with increasing pressure up to 350 MPa, which means that MC undergoes phase separation prior to gelation on heating under high pressure above 200 MPa. Similar results were obtained for the melting process of MC gel on cooling. The unique behavior of the sol-gel transition of MC under high pressure has been interpreted in terms of the destruction of hydrophobic hydration by compression.

  5. Non-toxic agarose/gelatin-based microencapsulation system containing gallic acid for antifungal application.

    PubMed

    Lam, P-L; Gambari, R; Kok, S H-L; Lam, K-H; Tang, J C-O; Bian, Z-X; Lee, K K-H; Chui, C-H

    2015-02-01

    Aspergillus niger (A. niger) is a common species of Aspergillus molds. Cutaneous aspergillosis usually occurs in skin sites near intravenous injection and approximately 6% of cutaneous aspergillosis cases which do not involve burn or HIV-infected patients are caused by A. niger. Biomaterials and biopharmaceuticals produced from microparticle-based drug delivery systems have received much attention as microencapsulated drugs offer an improvement in therapeutic efficacy due to better human absorption. The frequently used crosslinker, glutaraldehyde, in gelatin-based microencapsulation systems is considered harmful to human beings. In order to tackle the potential risks, agarose has become an alternative polymer to be used with gelatin as wall matrix materials of microcapsules. In the present study, we report the eco-friendly use of an agarose/gelatin-based microencapsulation system to enhance the antifungal activity of gallic acid and reduce its potential cytotoxic effects towards human skin keratinocytes. We used optimal parameter combinations, such as an agarose/gelatin ratio of 1:1, a polymer/oil ratio of 1:60, a surfactant volume of 1% w/w and a stirring speed of 900 rpm. The minimum inhibitory concentration of microencapsulated gallic acid (62.5 µg/ml) was significantly improved when compared with that of the original drug (>750 µg/ml). The anti-A. niger activity of gallic acid -containing microcapsules was much stronger than that of the original drug. Following 48 h of treatment, skin cell survival was approximately 90% with agarose/gelatin microcapsules containing gallic acid, whereas cell viability was only 25-35% with free gallic acid. Our results demonstrate that agarose/gelatin-based microcapsules containing gallic acid may prove to be helpful in the treatment of A. niger-induced skin infections near intravenous injection sites.

  6. Synthesis of an imprinted hybrid organic-inorganic polymeric sol-gel matrix toward the specific binding and isotherm kinetics investigation of creatinine.

    PubMed

    Chang, Yong-Sheun; Ko, Ting-Hsien; Hsu, Ting-Jung; Syu, Mei-Jywan

    2009-03-15

    Hybrid organic-inorganic polymeric sol-gel materials imprinted with creatinine template molecules were synthesized for the specific binding of creatinine. Creatinine is a metabolite from creatine and is the final product from kidney metabolism. Therefore, creatinine can be an important index to estimate the function of the kidney. It was then chosen as the target molecule in this work. To achieve the specific binding toward creatinine, molecular imprinting was used to create a polymeric matrix for the regarding purpose. Sol-gel was further added to create a rigid network structure for the absorption of creatinine. An inorganic precursor, tetraethoxysilane (TEOS), was mixed with an organic functional monomer, 2-acrylamido-2-methylpropane-sulfonic acid (AMPS), and the creatinine template to form a hybrid organic-inorganic imprinted polymer. The chemical functionality was achieved as well as a confined matrix via the polymerization and the hydrolysis-condensation of the sol-gel. The imprinting effect from the hybrid materials against the corresponding nonimprinted was investigated. BET (Brunauer-Emmett-Teller) analysis was carried out for the imprinted and the nonimprinted materials. The specificity of the hybrid materials was further examined by capping the surface silanol groups with chloro-trimethylsilane (CTMS) and 1,1,1,3,3,3-hexamethyldisilazane (HMDS), respectively. The capping effect was compared and discussed from the binding results. Selectivity of the materials toward creatinine was obtained using mixture solutions in the presence of creatinine and its analogues. Reutilization and storage stability of the hybrid organic-inorganic imprinted material were also studied. Additionally, the affinity distribution of the hybrid imprinted materials derived from the allosteric model was also analyzed from the adsorption isotherm data.

  7. Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device.

    PubMed

    Oakley, Jennifer A; Shaw, Kirsty J; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-06-07

    A silica monolith used to support both electro-osmotic pumping (EOP) and the extraction/elution of DNA coupled with gel-supported reagents is described. The benefits of the combined EOP extraction/elution system were illustrated by combining DNA extraction and gene amplification using the polymerase chain reaction (PCR) process. All the reagents necessary for both processes were supported within pre-loaded gels that allow the reagents to be stored at 4 degrees C for up to four weeks in the microfluidic device. When carrying out an analysis the crude sample only needed to be hydrodynamically introduced into the device which was connected to an external computer controlled power supply via platinum wire electrodes. DNA was extracted with 65% efficiency after loading lysed cells onto a silica monolith. Ethanol contained within an agarose gel matrix was then used to wash unwanted debris away from the sample by EOP (100 V cm(-1) for 5 min). The retained DNA was subsequently eluted from the monolith by water contained in a second agarose gel, again by EOP using an electric field of 100 V cm(-1) for 5 min, and transferred into the PCR reagent containing gel. The eluted DNA in solution was successfully amplified by PCR, confirming that the concept of a complete self-contained microfluidic device could be realised for DNA sample clean up and amplification, using a simple pumping and on-chip reagent storage methodology.

  8. Agarose particle-templated porous bacterial cellulose and its application in cartilage growth in vitro.

    PubMed

    Yin, Na; Stilwell, Matthew D; Santos, Thiago M A; Wang, Huaping; Weibel, Douglas B

    2015-01-01

    Bacterial cellulose (BC) is a biocompatible hydrogel with a three-dimensional (3-D) structure formed by a dense network of cellulose nanofibers. A limitation of using BC for applications in tissue engineering is that the pore size of the material (∼0.02-10μm) is smaller than the dimensions of mammalian cells and prevents cells from penetrating into the material and growing into 3-D structures that mimic tissues. This paper describes a new route to porous bacterial cellulose (pBC) scaffolds by cultivating Acetobacter xylinum in the presence of agarose microparticles deposited on the surface of a growing BC pellicle. Monodisperse agarose microparticles with a diameter of 300-500μm were created using a microfluidic technique, layered on growing BC pellicles and incorporated into the polymer as A. xylinum cells moved upward through the growing pellicle. Removing the agarose microparticles by autoclaving produced BC gels containing a continuous, interconnected network of pores with diameters ranging from 300 to 500μm. Human P1 chondrocytes seeded on the scaffolds, replicated, invaded the 3-D porous network and distributed evenly throughout the substrate. Chondrocytes grown on pBC substrates displayed a higher viability compared to growth on the surface of unmodified BC substrates. The approach described in this paper introduces a new method for creating pBC substrates with user-defined control over the physical dimensions of the pore network, and demonstrates the application of these materials for tissue engineering.

  9. Oxidized dextrins as alternative crosslinking agents for polysaccharides: application to hydrogels of agarose-chitosan.

    PubMed

    Gómez-Mascaraque, Laura G; Méndez, José Alberto; Fernández-Gutiérrez, Mar; Vázquez, Blanca; San Román, Julio

    2014-02-01

    Hydrogel networks that combine suitable physical and biomechanical characteristics for tissue engineering scaffolds are in demand. The aim of this work was the development of hydrogel networks based on agarose and chitosan using oxidized dextrins as low cytotoxicity crosslinking agents, paying special attention to the study of the influence of the polysaccharide composition and oxidation degree of the dextrins in the final characteristics of the network. The results show that the formation of an interpenetrating or a semi-interpenetrating polymer network was mainly dependent on a minimum agarose content and degree of oxidation of dextrin. Spectroscopic, thermal and swelling analysis revealed good compatibility with an absence of phase separation of polysaccharides at agarose:chitosan proportions of 50:50 and 25:75. The analysis of atomic force microscopy images showed the formation of a fibrillar microstructure whose distribution within the crosslinked chitosan depended mainly on the crosslinker. All materials exhibited the viscoelastic behaviour typical of gels, with a constant storage modulus independent of frequency for all compositions. The stiffness was strongly influenced by the degree of oxidation of the crosslinker. Cellular response to the hydrogels was studied with cells of different strains, and cell adhesion and proliferation was correlated with the homogeneity of the samples and their elastic properties. Some hydrogel formulations seemed to be candidates for tissue engineering applications such as wound healing or soft tissue regeneration.

  10. Hierarchically designed agarose and poly(ethylene glycol) interpenetrating network hydrogels for cartilage tissue engineering.

    PubMed

    DeKosky, Brandon J; Dormer, Nathan H; Ingavle, Ganesh C; Roatch, Christopher H; Lomakin, Joseph; Detamore, Michael S; Gehrke, Stevin H

    2010-12-01

    A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials-agarose and poly(ethylene glycol) (PEG) diacrylate-were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

  11. A new nano-optical sensor thin film cadmium sulfide doped in sol-gel matrix for assessment of α-amylase activity in human saliva.

    PubMed

    Attia, M S; Zoulghena, H; Abdel-Mottaleb, M S A

    2014-02-21

    A novel, simple, sensitive and precise spectrofluorimetric method is developed for measuring the activity of the α-amylase enzyme in human saliva. The remarkable quenching of the luminescence intensity at 634 nm of nano CdS doped in a sol-gel matrix by various concentrations of maltose (produced from the reaction of the enzyme with the starch substrate) was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 4.8 × 10(-10) to 1.2 × 10(-5) mol L(-1) maltose with a correlation coefficient of 0.999 and a detection limit of 5.7 × 10(-11) mol L(-1). The method was used satisfactorily for assessment of the α-amylase activity in a number of human saliva samples collected from various healthy volunteers.

  12. Inhibition of vimentin or B1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

    SciTech Connect

    Zhang, Xueping; Fournier, Marcia V; Ware, Joy L; Bissell, Mina J; Yacoub, Adly; Zehner, Zendra E

    2008-06-12

    Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin, or {alpha}6 and {beta}1 integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via small interfering RNA interference or the expression of {alpha}6 and {beta}1 integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and {beta}1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis. [Mol Cancer Ther 2009;8(3):499-508].

  13. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    PubMed

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.

  14. Agarose encapsulated mesoporous carbonated hydroxyapatite nanocomposites powder for drug delivery.

    PubMed

    Kolanthai, Elayaraja; Abinaya Sindu, P; Thanigai Arul, K; Sarath Chandra, V; Manikandan, E; Narayana Kalkura, S

    2017-01-01

    The powder composites are predominantly used for filling of voids in bone and as drug delivery carrier to prevent the infection or inflammatory reaction in the damaged tissues. The objective of this work was to study the synthesis of agarose encapsulation on carbonated hydroxyapatite powder and their biological and drug delivery properties. Mesoporous, nanosized carbonated hydroxyapatite/agarose (CHAp/agarose) powder composites were prepared by solvothermal method and subsequently calcined to study the physico-chemical changes, if it subjected to thermal exposure. The phase of the as-synthesized powder was CHAp/agarose whereas the calcinated samples were non-stoichiometric HAp. The CHAp/agarose nanorods were of length 10-80nm and width 40-190nm for the samples synthesized at temperatures 120°C (ST120) and 150°C (ST150). The calcination process produced spheres (10-50nm) and rods with reduced size (40-120nm length and 20-30nm width). Composites were partially dissolved in SBF solution followed by exhibited better bioactivity than non-stoichiometric HAp confirmed by gravimetric method. Hemo and biocompatibility remained unaffected by presence of agarose or carbonate in the HAp. Specific surface area of the composites was high and exhibited an enhanced amoxicillin and 5-fluorouracil release than the calcined samples. The composites demonstrated a strong antimicrobial activity against E. coli, S. aureus and S. epidermidis. The ST120 showed prolonged drug (AMX and 5-Fcil) release and antimicrobial efficacy than ST150 and calcined samples. This technique would be simple and rapid for composites preparation, to produce high quality crystalline, resorbable, mesoporous and bioactive nanocomposite (CHAp/agarose) powders. This work provides new insight into the role of agarose coated on bioceramics by solvothermal technique and suggests that CHAp/agarose composites powders are promising materials for filling of void in bone and drug delivery applications.

  15. Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

    PubMed Central

    Pabst, Breana; Pitts, Betsey; Lauchnor, Ellen

    2016-01-01

    An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrient- and oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h−1) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 μm. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance. PMID:27503656

  16. Gel Electrophoresis--The Easy Way for Students

    ERIC Educational Resources Information Center

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  17. Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

    PubMed

    Davey, M W; Sulkowski, E; Carter, W A

    1976-02-10

    Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside

  18. Novel hydrophobic interaction chromatography matrix for specific isolation and simple elution of immunoglobulins (A, G, and M) from porcine serum.

    PubMed

    Ramos-Clamont, Gabriela; del Carmen Candia-Plata, Maria; Zamudio, Roberto Guzman; Vazquez-Moreno, Luz

    2006-07-28

    A new, highly acetylated agarose matrix (HA-Sepharose) was synthesized and used as a hydrophobic interaction chromatography (HIC) medium to specifically isolate immunoglobulins (Igs) from porcine serum. Recovery of Igs was in a single step and under mild conditions. HA-Sepharose adsorption was studied in terms of salt, gel acetylation time, flow rate, and protein concentration on the loading buffer. At 0.5 M Na2SO4, control with unmodified Sepharose retained a small fraction (0.70 mg/mL of matrix) of serum albumin. On the contrary HA-Sepharose retained primary Igs (IgA, IgG, and 53% of IgM) as revealed by sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS-PAGE), quantitative radial immunodiffusion and immunodetection. At a flow rate of 1 mL/min, the HA-Sepharose column capacity (3.9 mg/mL of matrix) was similar to the reported capacity for the commercial thiophilic T-gel. However, HA-Sepharose showed higher recovery of IgA and IgM than the T-gel in the same salt conditions, clearly an advantage in terms of immunoglobulin recovery strategies. Acetylation changed the matrix adsorption from albumin to immunoglobulins; thus, the highly acetylated gel rendered recoveries of Igs from unprocessed porcine serum practically free of albumin.

  19. A detailed study of homogeneous agarose/hydroxyapatite nanocomposites for load-bearing bone tissue.

    PubMed

    Hu, Jingxiao; Zhu, Youjia; Tong, Hua; Shen, Xinyu; Chen, Li; Ran, Jiabing

    2016-01-01

    Agarose/hydroxyapatite (agar/HA) nanocomposites for load-bearing bone substitutes were successfully fabricated via a novel in situ precipitation method. Observation via SEM and TEM revealed that the spherical inorganic nanoparticles of approximately 50 nm were well dispersed in the organic matrix, and the crystallographic area combined closely with the amorphous area. The uniform dispersion of HA nanoparticles had prominent effect on improving the mechanical properties of the agar/HA nanocomposites (the highest elastic modulus: 1104.42 MPa; the highest compressive strength: 400.039 MPa), which proved to be potential load-bearing bone substitutes. The thermal stability of agarose and nanocomposites was also studied. The MG63 osteoblast-like cells on the composite disks displayed fusiform and polygonal morphology in the presence of HA, suggesting that the cell maturation was promoted. The results of cell proliferation and cell differentiation indicated that the cells cultured on the agar/HA composite disks significantly increased the alkaline phosphatase activity and calcium deposition. The structural role of agarose in the composite system was investigated to better understand the effect of biopolymer on structure and properties of the composites. The optimal properties were the result of a comprehensive synergy of the components.

  20. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    SciTech Connect

    Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

    1987-09-04

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

  1. Mesoporous Silica Gel-Based Mixed Matrix Membranes for Improving Mass Transfer in Forward Osmosis: Effect of Pore Size of Filler

    NASA Astrophysics Data System (ADS)

    Lee, Jian-Yuan; Wang, Yining; Tang, Chuyang Y.; Huo, Fengwei

    2015-11-01

    The efficiency of forward osmosis (FO) process is generally limited by the internal concentration polarization (ICP) of solutes inside its porous substrate. In this study, mesoporous silica gel (SG) with nominal pore size ranging from 4-30 nm was used as fillers to prepare SG-based mixed matrix substrates. The resulting mixed matrix membranes had significantly reduced structural parameter and enhanced membrane water permeability as a result of the improved surface porosity of the substrates. An optimal filler pore size of ~9 nm was observed. This is in direct contrast to the case of thin film nanocomposite membranes, where microporous nanoparticle fillers are loaded to the membrane rejection layer and are designed in such a way that these fillers are able to retain solutes while allowing water to permeate through them. In the current study, the mesoporous fillers are designed as channels to both water and solute molecules. FO performance was enhanced at increasing filler pore size up to 9 nm due to the lower hydraulic resistance of the fillers. Nevertheless, further increasing filler pore size to 30 nm was accompanied with reduced FO efficiency, which can be attributed to the intrusion of polymer dope into the filler pores.

  2. Effect of Rheology and Poloxamers Properties on Release of Drugs from Silicon Dioxide Gel-Filled Hard Gelatin Capsules-A Further Enhancement of Viability of Liquid Semisolid Matrix Technology.

    PubMed

    Sultana, Misbah; Butt, Mobashar Ahmad; Saeed, Tariq; Mahmood, Rizwan; Ul Hassan, Saeed; Hussain, Khalid; Raza, Syed Atif; Ahsan, Muhammad; Bukhari, Nadeem Irfan

    2016-12-08

    The liquid and semisolid matrix technology, filling liquids, semi-solids and gels in hard gelatin capsule are promising, thus, there is a need of enhanced research interest in the technology. Therefore, the present study was aimed to investigate isoniazid (freely soluble) and metronidazole (slightly soluble) gels filled in hard gelatin capsules for the effect of poloxamers of different viscosities on release of the drugs. Gel of each drug (10% w/w, particle size 180-250 μm), prepared by mixing poloxamer and 8% w/w hydrophilic silicon dioxide (Aerosil® A200), was assessed for rheology, dispersion stability and release profile. Both the drugs remained dispersed in majority of gels for more than 30 days, and dispersions were depended on gels' viscosity, which was further depended on viscosity of poloxamers. A small change in viscosity was noted in gels on storage. FTIR spectra indicated no interactions between components of the gels. The gels exhibited thixotropic and shear-thinning behaviour, which were suitable for filling in hard gelatin capsules without any leakage from the capsules. The release of both drugs from the phase-stable gels for 30 days followed first-order kinetics and was found to be correlated to drugs' solubility, poloxamers' viscosity, polyoxyethylene contents and proportion of block copolymer (poloxamers) in the gels. The findings of the present study indicated that release of drugs of different solubilities (isoniazid and metronidazole) might be modified from gels using different poloxamers and Aerosil® A200.

  3. Initial Fiber Alignment Pattern Alters Extracellular Matrix Synthesis in Fibroblast Populated Fibrin Gel Cruciforms and Correlates with Predicted Tension

    PubMed Central

    Sander, E.A.; Barocas, V.H.; Tranquillo, R.T.

    2013-01-01

    Human dermal fibroblasts entrapped in fibrin gels cast in cross-shaped (cruciform) geometries with 1:1 and 1:0.5 ratios of arm widths were studied to assess whether tension and alignment of the cells and fibrils affected ECM deposition. The cruciforms of contrasting geometry (symmetric vs. asymmetric), which developed different fiber alignment patterns, were harvested at 2, 5, and 10 weeks of culture. Cruciforms were subjected to planar biaxial testing, polarimetric imaging, DNA and biochemical analyses, histological staining, and SEM imaging. As the cruciforms compacted and developed fiber alignment, fibrin was degraded and elastin and collagen were produced in a geometry-dependent manner. Using a continuum mechanical model that accounts for direction-dependent stress due to cell traction forces and cell contact guidance with aligned fibers that occurs in the cruciforms, the mechanical stress environment was concluded to influence collagen deposition, with deposition being greatest in the narrow arms of the asymmetric cruciform where stress was predicted to be largest. PMID:21046467

  4. Interaction of electromagnetic fields with chondrocytes in gel culture. Final report, 15 Jan 92-14 Jan 94

    SciTech Connect

    Grodzinsky, A.J.; Buschmann, M.D.; Gluzband, Y.A.

    1992-01-14

    The specific objectives of this research period were: (1) to quantify the effect of applied electric fields on chondrocyte metabolism, using a range of stimulation frequencies and amplitudes; (2) to compare the chondrocyte biosynthetic response to applied fields at early times in agarose gel culture before an extracellular matrix has accumulated and at later times after significant deposition of matrix around and between the cells; and (3) to begin to interpret the biosynthetic response to applied fields in terms of models of physical mechanisms. The results of these studies suggest that electric fields applied to chondrocytes in agarose can modulate the synthesis of proteoglycans and protein constituents. Biosynthesis may be inhibited or stimulated depending on the amplitude of the applied current density. In addition, the presence of extracellular matrix may enhance the ability of normal chondrocytes and cells in intact cartilage to respond to electric fields, although the presence of matrix was not required for the stimulatory response to be observed with Swarm rat chondrosarcoma cells.

  5. Effects of Agar Gel Strength and Fat on Oral Breakdown, Volatile Release, and Sensory Perception Using in Vivo and in Vitro Systems.

    PubMed

    Frank, Damian; Eyres, Graham T; Piyasiri, Udayasika; Cochet-Broch, Maeva; Delahunty, Conor M; Lundin, Leif; Appelqvist, Ingrid M

    2015-10-21

    The density and composition of a food matrix affect the rates of oral breakdown and in-mouth flavor release as well as the overall sensory experience. Agar gels of increasing concentration (1.0, 1.7, 2.9, and 5% agarose) with and without added fat (0, 2, 5, and 10%) were spiked with seven aroma volatiles. Differences in oral processing and sensory perception were systematically measured by a trained panel using a discrete interval time intensity method. Volatile release was measured in vivo and in vitro by proton transfer reaction mass spectrometry. Greater oral processing was required as agar gel strength increased, and the intensity of flavor-related sensory attributes decreased. Volatile release was inversely related to gel strength, showing that physicochemical phenomena were the main mechanisms underlying the perceived sensory changes. Fat addition reduced the amount of oral processing and had differential effects on release, depending on the fat solubility or lipophilicity of the volatiles.

  6. Thermoreversible gelation in aqueous binary solvents of chemically modified agarose.

    PubMed

    Dahmani, Mohammed; Ramzi, Mohamed; Rochas, Cyrille; Guenet, Jean-Michel

    2003-01-15

    The thermoreversible gelation of chemically modified agarose has been studied in aqueous binary solvents (dimethyl sulfoxide and a series of formamide) by differential calorimetry, mechanical testing, and small-angle neutron scattering. The temperature-composition phase diagrams have been established. It is concluded that gelation is promoted by the formation of ternary complexes modified agarose/water/cosolvent, wherein the cosolvent mediates the interaction between chains through the formation of electrostatic interactions.

  7. A method for horizontal polyacrylamide slab gel electrophoresis.

    PubMed

    Bellomy, G R; Record, M T

    1989-01-01

    We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.

  8. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  9. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  10. A dual-ion imprinted polymer embedded in sol-gel matrix for the ultra trace simultaneous analysis of cadmium and copper.

    PubMed

    Bali Prasad, Bhim; Jauhari, Darshika; Verma, Archana

    2014-03-01

    In simultaneous determination of group of elements, there are inter-metallic interactions which result in a non-linear relationship between the peak current and ionic concentration for each of the element, at bare (unmodified) electrode. To resolve this problem, we have resorted, for the first time, to develop a modified pencil graphite electrode using a typical ion imprinted polymer network (dual-ion imprinted polymer embedded in sol-gel matrix (inorganic-organic hybrid nano-material)) for the simultaneous analysis of a binary mixture of Cd(II) and Cu(II) ions, without any complication of inter-metallic interactions and competitive bindings, in real samples. The adequate resolution of differential pulse anodic stripping voltammetry peaks by 725 mV (cf, 615 mV with unmodified electrode), without any cross-reactivity and the stringent detection limits as low as, 0.050 and 0.034 ng mL(-1) (S/N=3) for Cd(II) and Cu(II) ions, respectively by the proposed sensor can be considered useful for the primitive diagnosis of several chronic diseases in clinical settings.

  11. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    PubMed

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  12. In vivo bioengineered ovarian tumors based on collagen, matrigel, alginate and agarose hydrogels: a comparative study.

    PubMed

    Zheng, Li; Hu, Xuefeng; Huang, Yuanjie; Xu, Guojie; Yang, Jinsong; Li, Li

    2015-01-29

    Scaffold-based tumor engineering is rapidly evolving the study of cancer progression. However, the effects of scaffolds and environment on tumor formation have seldom been investigated. In this study, four types of injectable hydrogels, namely, collagen type I, Matrigel, alginate and agarose gels, were loaded with human ovarian cancer SKOV3 cells and then injected into nude mice subcutaneously. The growth of the tumors in vitro was also investigated. After four weeks, the specimens were harvested and analyzed. We found that tumor formation by SKOV3 cells was best supported by collagen, followed by Matrigel, alginate, control (without scaffold) and agarose in vivo. The collagen I group exhibited a larger tumor volume with increased neovascularization and increased necrosis compared with the other materials. Further, increased MMP activity, upregulated expression of laminin and fibronectin and higher levels of HIF-1α and VEGF-A in the collagen group revealed that the engineered tumor is closer to human ovarian carcinoma. In order, collagen, Matrigel, alginate, control (without scaffold) and agarose exhibited decreases in tumor formation. All evidence indicated that the in vivo engineered tumor is scaffold-dependent. Bioactive hydrogels are superior to inert hydrogels at promoting tumor regeneration. In particular, biomimetic hydrogels are advantageous because they provide a microenvironment that mimics the ECM of natural tumors. On the other hand, typical features of cancer cells and the expression of genes related to cancer malignancy were far less similar to the natural tumor in vitro, which indicated the importance of culture environment in vivo. Superior to the in vitro culture, nude mice can be considered satisfactory in vivo 'bioreactors' for the screening of favorable cell vehicles for tumor engineering in vitro.

  13. Functionalized Agarose Self-Healing Ionogels Suitable for Supercapacitors.

    PubMed

    Trivedi, Tushar J; Bhattacharjya, Dhrubajyoti; Yu, Jong-Sung; Kumar, Arvind

    2015-10-12

    Agarose has been functionalized (acetylated/carbanilated) in an ionic liquid (IL) medium of 1-butyl-3-methylimidazolium acetate at ambient conditions. The acetylated agarose showed a highly hydrophobic nature, whereas the carbanilated agarose could be dissolved in water as well as in the IL medium. Thermoreversible ionogels were obtained by cooling the IL sols of carbanilated agarose at room temperature. The ionogel prepared from a protic-aprotic mixed-IL system (1-butyl-3-methylimidazolium chloride and N-(2-hydroxyethyl)ammonium formate) demonstrated a superior self-healing property, as confirmed from rheological measurements. The superior self-healing property of such an ionogel has been attributed to the unique inter-intra hydrogen-bonding network of functional groups inserted in the agarose. The ionogel was tested as a flexible solid electrolyte for an activated-carbon-based supercapacitor cell. The measured specific capacitance was found to be comparable with that of a liquid electrolyte system at room temperature and was maintained for up to 1000 charge-discharge cycles. Such novel functionalized-biopolymer self-healing ionogels with flexibility and good conductivity are desirable for energy-storage devices and electronic skins with superior lifespans and robustness.

  14. A self-assembling matrix-forming gel can be easily and safely applied to prevent delayed bleeding after endoscopic resections

    PubMed Central

    Pioche, Mathieu; Camus, Marine; Rivory, Jérôme; Leblanc, Sarah; Lienhart, Isabelle; Barret, Maximilien; Chaussade, Stanislas; Saurin, Jean-Christophe; Prat, Frederic; Ponchon, Thierry

    2016-01-01

    Background: Endoscopic resections have low morbidity and mortality. Delayed bleeding has been reported in approximately 1 – 15 % of cases, increasing with antiplatelet/anticoagulant therapy or portal hypertension. A self-assembling peptide (SAP) forming a gel could protect the mucosal defect during early healing. This retrospective trial aimed to assess the safety and efficacy of SAP in preventing delayed bleeding after endoscopic resections. Methods: Consecutive patients with endoscopic resections were enrolled in two tertiary referral centers. Patients with a high risk of bleeding (antiplatelet agents, anticoagulation drugs with heparin bridge therapy, and cirrhosis with portal hypertension) were also included. The SAP gel was applied immediately after resection to cover the whole ulcer bed. Results: In total, 56 patients were included with 65 lesions (esophagus [n = 8], stomach [n = 22], duodenum [n = 10], ampullary [n = 3], colon [n = 7], and rectum [n = 15]) in two centers. Among those 65 lesions, 29 were resected in high risk situations (9 uninterrupted aspirin therapy, 6 heparin bridge therapies, 5 cirrhosis and portal hypertension, 1 both cirrhosis and heparin bridge, 3 both cirrhosis and uninterrupted aspirin, 3 large duodenal lesions > 2 cm, and 2 early introduction of clopidogrel at day 1). The resection technique was endoscopic submucosal dissection (ESD) in 40 cases, en bloc endoscopic mucosal resection (EMR) in 16, piecemeal EMR in 6, and ampullectomy in 3. The mean lesion size was 37.9 mm (SD: 2.2 mm) with a mean area of 6.3 cm2 (SD: 3.5 cm2). No difficulty was noted during application. Four delayed overt bleedings occurred (6.2 %) (3 hematochezia, 1 hematemesis) requiring endoscopic hemostasis. The mean hemoglobin drop off was 0.6 g/dL (– 0.6 to 3.1 g/dL). No adverse events occurred. Conclusion: The use of this novel extracellular matrix scaffold may help to reduce post-endoscopic resection

  15. Matrix effects on the photocatalytic oxidation of alcohols by [nBu4N]4W10O32 incorporated into sol-gel silica.

    PubMed

    Molinari, Alessandra; Bratovcic, Amra; Magnacca, Giuliana; Maldotti, Andrea

    2010-09-07

    Two heterogeneous photocatalysts have been prepared by entrapment of [nBu(4)N](4)W(10)O(32) in a silica matrix, through a sol-gel procedure: SiO(2)/W30% and SiO(2)/W10% with 30% and 10% of decatungstate, respectively. They are characterized by the presence of micropores of about 7 A and 15 A and mesopores of about 25 A. Due to different preparation procedures, SiO(2)/W10% presents a more remarkable porous network than SiO(2)/W30%. The morphological features of SiO(2)/W30% and SiO(2)/W10% differ from those of their parent material SiO(2)/W0%, indicating that incorporation of the decatungstate induces a significant modification of the porous texture of the siliceous material. These photocatalysts demonstrate good stability in the oxygen-assisted photooxidation of 1-pentanol and 3-pentanol, which have been chosen as models of primary and secondary aliphatic alcohols. In particular, photoexcitation (lambda > 290 nm, 25 degrees C, 760 torr of O(2)) leads to conversion of these two substrates to pentanal or 3-pentanone, with a mass balance of about 90%. There is a strong effect of the solid support on the reactivity of the two alcoholic substrates. In particular, oxidation of 1-pentanol with SiO(2)/W10% is about four times faster than with [nBu(4)N](4)W(10)O(32) in homogeneous solution. Preferential adsorption phenomena, due to the hydrophilic character of silica explain the photocatalytic properties of the two heterogeneous systems, because adsorption favours the contact between the photoexcited decatungstate and the primary OH group of 1-pentanol. Moreover, some kind of shape selectivity, due to the microporous structure of the investigated materials, likely contributes to control the conversion yields.

  16. Characterization of crystals of the Hjc resolvase from Archaeoglobus fulgidus grown in gel by counter-diffusion

    SciTech Connect

    Biertümpfel, Christian; Basquin, Jérôme; Birkenbihl, Rainer P.; Suck, Dietrich; Sauter, Claude

    2005-07-01

    The Holliday junction-cutting enzyme Hjc from A. fulgidus was crystallized by the counter-diffusion method in agarose gel and complete data were collected at 2.7 Å resolution using synchrotron radiation. Holliday junction-resolving enzymes are ubiquitous proteins that play a key role in DNA repair and reorganization by homologous recombination. The Holliday junction-cutting enzyme (Hjc) from the archaeon Archaeoglobus fulgidus is a member of this group. The first Hjc crystals were obtained by conventional sparse-matrix screening. They exhibited an unusually elongated unit cell and their X-ray characterization required special care to avoid spot overlaps along the c* axis. The use of an arc appended to the goniometric head allowed proper orientatation of plate-like crystals grown in agarose gel by counter-diffusion. Thus, complete diffraction data were collected at 2.7 Å resolution using synchrotron radiation. They belong to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 37.4, c = 271.8 Å.

  17. Pulsed-field gel electrophoresis of bacterial chromosomes.

    PubMed

    Mawer, Julia S P; Leach, David R F

    2013-01-01

    The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

  18. Quaternary ammonium substituted agarose as surface coating for capillary electrophoresis.

    PubMed

    Ullsten, Sara; Söderberg, Lennart; Folestad, Staffan; Markides, Karin E

    2004-05-01

    A novel positively charged polymer of quaternary ammonium substituted agarose (Q-agarose) has been synthesized and explored for use as a coating in capillary electrophoresis. The fast and simple coating procedure is based on a multi-site electrostatic interaction between the polycationic agarose polymer and the negatively charged fused-silica surface. By simply flushing fused-silica capillaries with hot polymer solution a positively charged, hydrophilic deactivation layer is achieved. The polymer surface provides an intermediate electroosmotic flow of reversed direction, over a range of pH 2-11, compared to unmodified fused-silica. The coating procedure was highly reproducible with an RSD of 4%, evaluated as the electroosmotic flow mobility for 30 capillaries prepared at 10 different occasions. The application of Q-agarose coated capillaries in separation science was investigated using a set of basic drugs and model proteins and peptides. Due to the intermediate electroosmotic flow generated, the resolution of basic drugs could be increased, compared to using bare fused-silica capillaries. Moreover, the coating enabled separation of proteins and peptides with efficiencies up to 300.000 plates m(-1).

  19. Sol-Gel Glasses

    NASA Technical Reports Server (NTRS)

    Mukherjee, S. P.

    1985-01-01

    Multicomponent homogeneous, ultrapure noncrystalline gels/gel derived glasses are promising batch materials for the containerless glass melting experiments in microgravity. Hence, ultrapure, homogeneous gel precursors could be used to: (1) investigate the effect of the container induced nucleation on the glass forming ability of marginally glass forming compositions; and (2) investigate the influence of gravity on the phase separation and coarsening behavior of gel derived glasses in the liquid-liquid immiscibility zone of the nonsilicate systems having a high density phase. The structure and crystallization behavior of gels in the SiO2-GeO2 as a function of gel chemistry and thermal treatment were investigated. As are the chemical principles involved in the distribution of a second network former in silica gel matrix being investigated. The procedures for synthesizing noncrystalline gels/gel-monoliths in the SiO2-GeO2, GeO2-PbO systems were developed. Preliminary investigations on the levitation and thermal treatment of germania silicate gel-monoliths in the Pressure Facility Acoustic Levitator were done.

  20. High-throughput tracking of single yeast cells in a microfluidic imaging matrix.

    PubMed

    Falconnet, D; Niemistö, A; Taylor, R J; Ricicova, M; Galitski, T; Shmulevich, I; Hansen, C L

    2011-02-07

    Time-lapse live cell imaging is a powerful tool for studying signaling network dynamics and complexity and is uniquely suited to single cell studies of response dynamics, noise, and heritable differences. Although conventional imaging formats have the temporal and spatial resolution needed for such studies, they do not provide the simultaneous advantages of cell tracking, experimental throughput, and precise chemical control. This is particularly problematic for system-level studies using non-adherent model organisms such as yeast, where the motion of cells complicates tracking and where large-scale analysis under a variety of genetic and chemical perturbations is desired. We present here a high-throughput microfluidic imaging system capable of tracking single cells over multiple generations in 128 simultaneous experiments with programmable and precise chemical control. High-resolution imaging and robust cell tracking are achieved through immobilization of yeast cells using a combination of mechanical clamping and polymerization in an agarose gel. The channel and valve architecture of our device allows for the formation of a matrix of 128 integrated agarose gel pads, each allowing for an independent imaging experiment with fully programmable medium exchange via diffusion. We demonstrate our system in the combinatorial and quantitative analysis of the yeast pheromone signaling response across 8 genotypes and 16 conditions, and show that lineage-dependent effects contribute to observed variability at stimulation conditions near the critical threshold for cellular decision making.

  1. The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production.

    PubMed

    Kim, Hee Taek; Lee, Saeyoung; Kim, Kyoung Heon; Choi, In-Geol

    2012-03-01

    A sugar platform equipped with acetic acid, multiple agarases and neoagarobiose hydrolase (NABH) converted recalcitrant agar polysaccharide into monosugars, which was evaluated by simultaneous saccharification and fermentation (SSF). The sugar platform was divided into chemical liquefaction and enzymatic saccharification. The chemical liquefaction was carried out in mild conditions (using a dilute acetic acid at 80°C for 1-6h) to avoid the production of fermentation inhibitors and hence the highest degree of liquefaction of 95.6% (w/w) was obtained. We mimicked the natural agarolytic pathway using three microbial agarases (Aga16B, Aga50D and DagA) and NABH, and the enzyme system converted 79.1% of agarose to monosugars. The chemical liquefaction and SSF of 30 g/l agarose resulted in 4.4 g/l ethanol concentration and 49.3% of the theoretical ethanol yield to d-galactose. This is the first report on the complete enzymatic conversion of agarose into its monosugars and the SSF of agarose into ethanol.

  2. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems.

  3. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    SciTech Connect

    Zhang, J.S.; Giometti, C.S.; Tollaksen, S.L.

    1989-04-25

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower and of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  4. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    SciTech Connect

    Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

    1989-01-01

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  5. Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration

    NASA Astrophysics Data System (ADS)

    Carriel, Víctor; Garrido-Gómez, Juan; Hernández-Cortés, Pedro; Garzón, Ingrid; García-García, Salomé; Sáez-Moreno, José Antonio; Sánchez-Quevedo, María del Carmen; Campos, Antonio; Alaminos, Miguel

    2013-04-01

    Objective. The objective was to study the effectiveness of a commercially available collagen conduit filled with fibrin-agarose hydrogels alone or with fibrin-agarose hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs) in a rat sciatic nerve injury model. Approach. A 10 mm gap was created in the sciatic nerve of 48 rats and repaired using saline-filled collagen conduits or collagen conduits filled with fibrin-agarose hydrogels alone (acellular conduits) or with hydrogels containing ADMSCs (ADMSC conduits). Nerve regeneration was assessed in clinical, electrophysiological and histological studies. Main results. Clinical and electrophysiological outcomes were more favorable with ADMSC conduits than with the acellular or saline conduits, evidencing a significant recovery of sensory and motor functions. Histological analysis showed that ADMSC conduits produce more effective nerve regeneration by Schwann cells, with higher remyelination and properly oriented axonal growth that reached the distal areas of the grafted conduits, and with intensely positive expressions of S100, neurofilament and laminin. Extracellular matrix was also more abundant and better organized around regenerated nerve tissues with ADMSC conduits than those with acellular or saline conduits. Significance. Clinical, electrophysiological and histological improvements obtained with tissue-engineered ADMSC conduits may contribute to enhancing axonal regeneration by Schwann cells.

  6. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    PubMed

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution.

  7. Enzymatic production of 3,6-anhydro-L-galactose from agarose and its purification and in vitro skin whitening and anti-inflammatory activities.

    PubMed

    Yun, Eun Ju; Lee, Saeyoung; Kim, Ji Hye; Kim, Bo Bae; Kim, Hee Taek; Lee, Sun Hee; Pelton, Jeffrey G; Kang, Nam Joo; Choi, In-Geol; Kim, Kyoung Heon

    2013-04-01

    3,6-Anhydro-L-galactose (L-AHG) constitutes 50% of agarose, which is the main component of red macroalgae. No information is currently available on the mass production, metabolic fate, or physiological effects of L-AHG. Here, agarose was converted to L-AHG in the following three steps: pre-hydrolysis of agarose into agaro-oligosaccharides by using acetic acid, hydrolysis of the agaro-oligosaccharides into neoagarobiose by an exo-agarase, and hydrolysis of neoagarobiose into L-AHG and galactose by a neoagarobiose hydrolase. After these three steps, L-AHG was purified by adsorption and gel permeation chromatographies. The final product obtained was 95.6% pure L-AHG at a final yield of 4.0% based on the initial agarose. In a cell proliferation assay, L-AHG at a concentration of 100 or 200 μg/ mL did not exhibit any significant cytotoxicity. In a skin whitening assay, 100 μg/ mL of L-AHG showed significantly lower melanin production compared to arbutin. L-AHG at 100 and 200 μg/ mL showed strong anti-inflammatory activity, indicating the significant suppression of nitrite production. This is the first report on the production of high-purity L-AHG and its physiological activities.

  8. Synthesis of agarose-graft-poly[3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate] zwitterionic graft copolymers via ATRP and their thermally-induced aggregation behavior in aqueous media.

    PubMed

    Tian, Miao; Wang, Jinmei; Zhang, Ershuai; Li, Junjie; Duan, Cuimi; Yao, Fanglian

    2013-06-25

    A novel polysaccharide-based zwitterionic copolymer, agarose-graft-poly[3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate] (agarose-g-PDMAPS) with UCST, depending both on hydrogen bonding and electrostatic interaction, was synthesized by ATRP, and its aggregation behavior in aqueous media was investigated in detail. Proton nuclear magnetic resonance spectroscopy, Fourier transform-infrared spectroscopy, and gel-permeation chromatography were performed to characterize the copolymer. Thermosensitive behaviors of the copolymers in water, NaCl, and urea solution were tracked by ultraviolet, dynamic light scattering, and transmission electron microscopy analysis. It was found that the copolymers existed as "core-shell" spheres at an elevated temperature, as a result of the self-assembly of the agarose backbones located in the "core" driven by hydrogen-bonding interactions. When the copolymer solution was cooled below UCST, the core-shell spheres began to aggregate because of the electrostatic interactions and collapse of PDMAPS side chains in the "shell" layer. UCST of the copolymer could be tuned in a wide range, depending on the chain lengths of PDMAPS. This is the first example to investigate the thermosensitivity, combining ionic interactions of the zwitterionic side chains with hydrogen bondings from the biocompatible agarose backbones. The synthetic strategy presented here can be employed in the preparation of other novel biomaterials from a variety of polysaccharides.

  9. Agarose and methylcellulose hydrogel blends for nerve regeneration applications

    NASA Astrophysics Data System (ADS)

    Martin, Benton C.; Minner, Eric J.; Wiseman, Sherri L.; Klank, Rebecca L.; Gilbert, Ryan J.

    2008-06-01

    Trauma sustained to the central nervous system is a debilitating problem for thousands of people worldwide. Neuronal regeneration within the central nervous system is hindered by several factors, making a multi-faceted approach necessary. Two factors contributing to injury are the irregular geometry of injured sites and the absence of tissue to hold potential nerve guides and drug therapies. Biocompatible hydrogels, injectable at room temperature, that rapidly solidify at physiological temperatures (37 °C) are beneficial materials that could hold nerve guidance channels in place and be loaded with therapeutic agents to aid wound healing. Our studies have shown that thermoreversible methylcellulose can be combined with agarose to create hydrogel blends that accommodate these properties. Three separate novel hydrogel blends were created by mixing methylcellulose with one of the three different agaroses. Gelation time tests show that the blends solidify at a faster rate than base methylcellulose at 37 °C. Rheological data showed that the elastic modulus of the hydrogel blends rapidly increases at 37 °C. Culturing experiments reveal that the morphology of dissociated dorsal root ganglion neurons was not altered when the hydrogels were placed onto the cells. The different blends were further assessed using dissolution tests, pore size evaluations using scanning electron microscopy and measuring the force required for injection. This research demonstrates that blends of agarose and methylcellulose solidify much more quickly than plain methylcellulose, while solidifying at physiological temperatures where agarose cannot. These hydrogel blends, which solidify at physiological temperatures naturally, do not require ultraviolet light or synthetic chemical cross linkers to facilitate solidification. Thus, these hydrogel blends have potential use in delivering therapeutics and holding scaffolding in place within the nervous system.

  10. Gel-based optical waveguides with live cell encapsulation and integrated microfluidics.

    PubMed

    Jain, Aadhar; Yang, Allen H J; Erickson, David

    2012-05-01

    In this Letter, we demonstrate a biocompatible microscale optical device fabricated from agarose hydrogel that allows for encapsulation of cells inside an optical waveguide. This allows for better interaction between the light in the waveguide and biology, since it can interact with the direct optical mode rather than the evanescent field. We characterize the optical properties of the waveguide and further incorporate a microfluidic channel over the optical structure, thus developing an integrated optofluidic system fabricated entirely from agarose gel.

  11. BANANA GEL.

    PubMed

    McGuire, G; Falk, K G

    1922-03-20

    The conditions for the formation of gels from banana extracts were studied. Gels were obtained with extracts more alkaline than pH 7.0 with very small quantities of calcium, strontium, and barium salts, the gel formation with these salts decreasing in the indicated order. In solutions more acid than pH 6.0, no gels were obtained with these salts. Magnesium, lithium, and sodium salts did not cause gel formation either in acid or alkaline solutions. Pancreatine gave a gel on incubation with banana extract at pH 5.0. The gel-forming property of banana extracts was destroyed on boiling.

  12. Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

    PubMed Central

    McElfresh, Cameron; Wong, Lily R.

    2015-01-01

    Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. PMID:26070672

  13. Efficacy of transdermal magnesium ascorbyl phosphate delivery after ultrasound treatment with microbubbles in gel-type surrounding medium in mice.

    PubMed

    Liao, Ai-Ho; Lu, Ying-Jui; Hung, Chi-Ray; Yang, Meng-Yu

    2016-04-01

    Liquid microemulsions appropriate for topical application were obtained by increasing their viscosity through the addition of thickening agents. The present study first assessed the usefulness of ultrasound (US) plus US contrast agent, microbubbles (MBs), in agarose gel for enhancing transdermal drug delivery. The effect of US plus MBs in agarose gel on the penetration of the skin by magnesium ascorbyl phosphate (MAP) was explored both in vitro and in vivo. In the in vitro experiments, the stability of MBs was investigated by examining the penetration of MAP by the model drug, Evans blue, in two media: an agarose phantom and pig skin. The penetration depth in the agarose phantom and pig skin increased by 40% and 195%, respectively, when treated with US plus MBs in 0.1% agarose solution combined with MAP (UMB1), and by 48% and 206%, respectively, when treated with US plus MBs in 0.15% agarose solution and MAP (UMB2). The skin-whitening effects in C57BL/6J mice in the UMB1 and UMB2 groups over a 4-week experimental period were significantly increased by 63% and 70%, respectively, in the fourth week. The findings of this study suggest that the survival of MBs with US is affected by the viscosity of the surrounding medium, and that in mice, treatment with US plus MBs in a suitable agarose gel can increase skin permeability and enhance transdermal MAP delivery.

  14. Comparison of in-gel and on-membrane digestion methods at low to sub-pmol level for subsequent peptide and fragment-ion mass analysis using matrix-assisted laser-desorption/ionization mass spectrometry.

    PubMed

    Courchesne, P L; Luethy, R; Patterson, S D

    1997-01-01

    The success of the mass spectrometric-based approaches for the identification of gel-separated proteins relies upon recovery of peptides, without high levels of ionization-suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in-gel or on-membrane digestion provided a significant advantage when low to sub-pmol quantities of gel-separated proteins were analyzed by matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins of M(r) 17,000 to 97,000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in-gel digestion (using a microcolumn clean-up protocol, Courchesne, P.L. and Patterson, S. D., BioTechniques, 1997, in press) or on-membrane digestion following blotting to the PVDF-based membranes, Immobilon-P and Immobilon-CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon-P and Immobilon-CD, 0.5 pmol; and in-gel, 125 fmol), and searches of Swiss-Prot or a non-redundant database (> 193000 entries) successfully identified all of the proteins, except beta-casein. Fragment-ion spectra using a curved-field reflector MALDI-MS were obtained from more than one peptide per protein at loads down to 250 fmol (except beta-casein). Using the uninterpreted data, a search of the nonredundant database and a six-way translation of GenBank dbEST (> 2,208,000 entries total) was able to identify myoglobin, carbonic anhydrase II, and phosphorylase b.

  15. DNA electrophoresis in tri-block copolymer gels--experiments and Brownian dynamics simulation

    NASA Astrophysics Data System (ADS)

    Wei, Ling; van Winkle, David H.

    2015-03-01

    The mobility of double-stranded DNA ladders in Pluronics®P105, P123 and F127, was measured by two-dimensional gel electrophoresis. Pluronics®are triblock copolymers which form gel-like phases of micelles arranged with cubic order at room temperature. A 10 base pair and a 25 base pair DNA ladder were used as samples in gel electrophoresis. The monotonically decreasing mobility with increasing length observed in the agarose separations is not observed in separations in Pluronics®. Rather, a complicated dependence of mobility on DNA length is observed, where mobility vs. length increases for short DNA molecules then decreases for longer molecules. There is also a variation of mobility with length correlated to the micelle diameter. Brownian dynamics simulations of a discrete wormlike chain model were performed to simulate short DNA molecules migrating in free solution and in a face-centered cubic matrix. By incorporating hydrodynamic interactions, the trend of simulated length-dependent mobility qualitatively agrees with experimental measurements.

  16. Nanocrystal/sol-gel nanocomposites

    DOEpatents

    Petruska, Melissa A.; Klimov, Victor L.

    2007-06-05

    The present invention is directed to solid composites including colloidal nanocrystals within a sol-gel host or matrix and to processes of forming such solid composites. The present invention is further directed to alcohol soluble colloidal nanocrystals useful in formation of sol-gel based solid composites.

  17. Nanocrystal/sol-gel nanocomposites

    SciTech Connect

    Petruska, Melissa A; Klimov, Victor L

    2012-06-12

    The present invention is directed to solid composites including colloidal nanocrystals within a sol-gel host or matrix and to processes of forming such solid composites. The present invention is further directed to alcohol soluble colloidal nanocrystals useful in formation of sol-gel based solid composites

  18. Anion-switchable supramolecular gels for controlling pharmaceutical crystal growth

    NASA Astrophysics Data System (ADS)

    Foster, Jonathan A.; Piepenbrock, Marc-Oliver M.; Lloyd, Gareth O.; Clarke, Nigel; Howard, Judith A. K.; Steed, Jonathan W.

    2010-12-01

    We describe the use of low-molecular-weight supramolecular gels as media for the growth of molecular crystals. Growth of a range of crystals of organic compounds, including pharmaceuticals, was achieved in bis(urea) gels. Low-molecular-weight supramolecular gelators allow access to an unlimited range of solvent systems, in contrast to conventional aqueous gels such as gelatin and agarose. A detailed study of carbamazepine crystal growth in four different bis(urea) gelators, including a metallogelator, is reported. The crystallization of a range of other drug substances, namely sparfloxacin, piroxicam, theophylline, caffeine, ibuprofen, acetaminophen (paracetamol), sulindac and indomethacin, was also achieved in supramolecular gel media without co-crystal formation. In many cases, crystals can be conveniently recovered from the gels by using supramolecular anion-triggered gel dissolution; however, crystals of substances that themselves bind to anions are dissolved by them. Overall, supramolecular gel-phase crystallization offers an extremely versatile new tool in pharmaceutical polymorph screening.

  19. Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

    PubMed

    Becker, J Susanne; Mounicou, Sandra; Zoriy, Miroslav V; Becker, J Sabine; Lobinski, Ryszard

    2008-09-15

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.

  20. Identification of differentially expressed proteins of gamma-ray irradiated rat intestinal epithelial IEC-6 cells by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.

    PubMed

    Bo, Zhang; Yongping, Su; Fengchao, Wang; Guoping, Ai; Yongjiang, Wei

    2005-02-01

    To identify proteins involved in the processes of cellular and molecular response to radiation damage repair in intestinal epithelial IEC-6 cells, we comparatively analyzed the proteome of irradiated IEC-6 cells with that of normal cells. A series of methods were used, including two-dimensional gel electrophoresis (Z-DE), PDQuest software analysis of 2-DE gels, peptide mass fingerprinting based on matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), and Swiss-Prot database searching, to separate and identify differentially expressed proteins. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. Image analysis revealed that averages of 608 +/- 39 and 595 +/- 31 protein spots were detected in normal and irradiated IEC-6 cells, respectively. Sixteen differential protein spots were isolated from gels, and measured with MALDI-TOF-MS. A total of 14 spots yielded good spectra, and 11 spots matched with known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, and protein post-translational processes. Western blotting confirmed that stress-70 protein was down-regulated by gamma-irradiation. Up-regulation of ERP29 was confirmed by RT-PCR, indicating that it is involved in ionizing radiation. The clues provided by the comparative proteome strategy utilized here will shed light on molecular mechanisms of radiation damage repair in intestinal epithelial cells.

  1. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis.

    PubMed

    Huang, Qing; Fu, Wei-Ling

    2005-01-01

    Ethidium bromide (EB) is a mutagen and toxin that is widely used in the laboratory for visualization of nucleic acids. Safer nucleic acid stains, such as SYBR Gold, SYBR Green, GoldView, GeneFinder, and GoldStar, have been developed. However, there has been no systematic comparative analysis of the staining efficiencies of these dyes. In the present study, SYBR Gold, SYBR Green I, GoldView and EB were compared. Although both SYBR Gold and SYBR Green alter electrophoretic mobility and thus DNA size estimates, they are cost-effective alternatives to EB. SYBR Gold was more sensitive than SYBR Green I at detecting short fragments, but 50-bp bands were clearly visible using either dye when visualized with a long integration time. SYBR Gold or SYBR Green I are sensitive and relatively safe alternatives to EB. In our laboratory, the SYBR Gold method is now used routinely by all members of our group with great consistency and success.

  2. Novel spectrofluorimetric method for measuring the activity of the enzyme alpha-L-fucosidase using the nano composite optical sensor samarium(III)-doxycycline complex doped in sol-gel matrix.

    PubMed

    Attia, M S; Othman, A M; Aboaly, M M; Abdel-Mottaleb, M S A

    2010-07-15

    A novel, simple, sensitive, and precise spectrofluorimetric method was developed for measuring the activity of the enzyme alpha-L-fucosidase (AFU). The method was based upon measuring the quenching of the luminescence intensity of the produced yellow colored complex ion associate of 2-chloro-4-nitrophenol [2-CNP] and a nano composite optical sensor samarium(III)-doxycycline [Sm(3+)-DC](+) complex in a sol-gel matrix at 645 nm. The remarkable quenching of the luminescence intensity of the [Sm(3+)-DC](+) complex doped in a sol-gel matrix by various concentrations of the reagent [2-CNP] was successfully used as an optical sensor for the assessment of AFU activity. The calibration plot was achieved over the concentration range 3.4 x 10(-9)-1.0 x 10(-6) mol L(-1) [2-CNP] with a correlation coefficient of 0.99 and a detection limit of 6.0 x 10(-10) mol L(-1). The method was used satisfactorily for the assessment of the AFU activity in a number of serum samples collected from various patients. A significant correlation between the luminescence activity of the enzyme AFU measured by the proposed procedure and the standard method was applied to patients and controls. The method proceeds without practical artifacts compared to the standard method.

  3. Encapsulation of Polymer Colloids in a Sol-Gel Matrix. Direct-Writing of Coassembling Organic-Inorganic Hybrid Photonic Crystals.

    PubMed

    Mikosch, Annabel; Kuehne, Alexander J C

    2016-03-22

    The spontaneous self-assembly of polymer colloids into ordered arrangements provides a facile strategy for the creation of photonic crystals. However, these structures often suffer from defects and insufficient cohesion, which result in flaking and delamination from the substrate. A coassembly process has been developed for convective assembly, resulting in large-area encapsulated colloidal crystals. However, to generate patterns or discrete deposits in designated places, convective assembly is not suitable. Here we experimentally develop conditions for direct-writing of coassembling monodisperse dye-doped polystyrene particles with a sol-gel precursor to form solid encapsulated photonic crystals. In a simple procedure the colloids are formulated in a sol-gel precursor solution, drop-cast on a flat substrate, and dried. We here establish the optimal parameters to form reproducible highly ordered photonic crystals with good optical performance. The obtained photonic crystals interact with light in the visible spectrum with a narrow optical stop-gap.

  4. Coupling sodium dodecyl sulfate-capillary polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry via a poly(tetrafluoroethylene) membrane.

    PubMed

    Lu, Joann J; Zhu, Zaifang; Wang, Wei; Liu, Shaorong

    2011-03-01

    Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS-capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.

  5. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    PubMed

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  6. Immobilization of flax protoplasts in agarose and alginate beads. Correlation between ionically bound cell-wall proteins and morphogenetic response.

    PubMed Central

    Roger, D; David, A; David, H

    1996-01-01

    Linum usitatissimum protoplast-derived colonies that are cultured in auxin-supplemented medium and immobilized in Ca(2+)-alginate matrix form round colonies that develop into polarized, embryo-like structures. On the other hand, protoplast-derived colonies that are immobilized in agarose do not show an organized morphogenetic response, and unique, ionically bound cell-wall protein patterns match this response. Although only slight differences in neosynthesized or total constitutive polypeptides are observed, dramatic changes in ionically bound cell-wall proteins are seen. In protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium, several basic polypeptides were strongly induced and were found tightly bound to the cell wall. In contrast, these basic proteins were found only weakly bound to the walls of protoplasts grown on agarose-solidified, auxin-containing medium or on Ca(2+)-alginate-solidified, auxin-free medium, in which they were released into the medium. Our results suggest that plant cells can perceive and respond to the adjacent extracellular matrix, since we show that the growth of flax cells on Ca(2+)-alginate in the presence of auxin-containing medium may promote the binding of specific proteins to the walls. This establishes a direct correlation of an embryo-like morphogenesis with ionically bound cell-wall basic proteins in flax protoplasts grown on Ca(2+)-alginate-solidified, auxin-containing medium. PMID:8938417

  7. Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior.

    PubMed

    Sun, Xiao; Heckscher, Ellie S

    2016-11-26

    Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex, including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed(1). In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

  8. Gel polymer electrolytes for batteries

    DOEpatents

    Balsara, Nitash Pervez; Eitouni, Hany Basam; Gur, Ilan; Singh, Mohit; Hudson, William

    2014-11-18

    Nanostructured gel polymer electrolytes that have both high ionic conductivity and high mechanical strength are disclosed. The electrolytes have at least two domains--one domain contains an ionically-conductive gel polymer and the other domain contains a rigid polymer that provides structure for the electrolyte. The domains are formed by block copolymers. The first block provides a polymer matrix that may or may not be conductive on by itself, but that can soak up a liquid electrolyte, thereby making a gel. An exemplary nanostructured gel polymer electrolyte has an ionic conductivity of at least 1.times.10.sup.-4 S cm.sup.-1 at 25.degree. C.

  9. Efficient dual-wavelength excitation of Tb3+ emission in rare-earth doped KYF4 cubic nanocrystals dispersed in silica sol-gel matrix

    NASA Astrophysics Data System (ADS)

    del-Castillo, J.; Yanes, A. C.; Santana-Alonso, A.; Méndez-Ramos, J.

    2014-11-01

    Energy transfer from Ce3+ to Tb3+ ions under UV excitation, giving rise to visible emissions, is investigated in sol-gel derived transparent nano-glass-ceramics containing cubic KYF4 nanocrystals, for different doping concentrations of rare-earth ions. Moreover, visible emissions of Tb3+ are also obtained under near-infrared excitation through energy transfer from Yb3+ ions by means of cooperative up-conversion processes. Thus, Ce3+-Tb3+-Yb3+ doped nano-glass-ceramics can be activated in a dual-wavelength mode yielding efficient blue-green emissions of particular interest in photovoltaic silicon solar cells and white-light emitting diodes.

  10. Adsorption of human serum proteins onto TREN-agarose: purification of human IgG by negative chromatography.

    PubMed

    Bresolin, Igor Tadeu Lazzarotto; Borsoi-Ribeiro, Mariana; Caro, Juliana Rodrigues; dos Santos, Francine Petit; de Castro, Marina Polesi; Bueno, Sonia Maria Alves

    2009-01-01

    Tris(2-aminoethyl)amine (TREN) - a chelating agent used in IMAC - immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90-95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.

  11. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

    PubMed

    Ersson, Clara; Möller, Lennart

    2011-11-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.

  12. Growth in Agarose of Human Cells Infected with Cytomegalovirus

    PubMed Central

    Lang, David J.; Montagnier, Luc; Latarjet, Raymond

    1974-01-01

    After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation. Images PMID:4367907

  13. High-quality substrate for fluorescence enhancement using agarose-coated silica opal film.

    PubMed

    Xu, Ming; Li, Juan; Sun, Liguo; Zhao, Yuanjin; Xie, Zhuoying; Lv, Linli; Zhao, Xiangwei; Xiao, Pengfeng; Hu, Jing; Lv, Mei; Gu, Zhongze

    2010-08-01

    To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.

  14. Annealing behavior of silver, copper, and silver-copper nanoclusters in a silica matrix synthesized by the sol-gel technique

    NASA Astrophysics Data System (ADS)

    De, G.; Gusso, M.; Tapfer, L.; Catalano, M.; Gonella, F.; Mattei, G.; Mazzoldi, P.; Battaglin, G.

    1996-12-01

    Silver, copper, and mixed silver-copper nanocluster-doped silica thin layers were prepared by the sol-gel process. Samples were heat treated in different annealing atmospheres (air, argon, or 5%H2-95%N2) in the temperature range 500-1100 °C. Specimens were characterized by optical absorption spectroscopy, Rutherford backscattering spectrometry, x-ray diffraction, and transmission electron microscopy. Cluster growth and dissolution, as well as migration of metal atoms towards the sample surface, with a subsequent evaporation, were observed to occur at temperatures that depend on the annealing atmosphere. In the mixed silver-copper system, the formation of Ag-Cu phase-separated clusters was observed.

  15. Agarose-chitosan-C18 film micro-solid phase extraction combined with high performance liquid chromatography for the determination of phenanthrene and pyrene in chrysanthemum tea samples.

    PubMed

    Ng, Nyuk Ting; Sanagi, Mohd Marsin; Wan Ibrahim, Wan Nazihah; Wan Ibrahim, Wan Aini

    2017-05-01

    Agarose-chitosan-immobilized octadecylsilyl-silica (C18) film micro-solid phase extraction (μSPE) was developed and applied for the determination of phenanthrene (PHE) and pyrene (PYR) in chrysanthemum tea samples using high performance liquid chromatography-ultraviolet detection (HPLC-UV). The film of blended agarose and chitosan allows good dispersion of C18, prevents the leaching of C18 during application and enhances the film mechanical stability. Important μSPE parameters were optimized including amount of sorbent loading, extraction time, desorption solvent and desorption time. The matrix match calibration curves showed good linearity (r⩾0.994) over a concentration range of 1-500ppb. Under the optimized conditions, the proposed method showed good limits of detection (0.549-0.673ppb), good analyte recoveries (100.8-105.99%) and good reproducibilities (RSDs⩽13.53%, n=3) with preconcentration factors of 4 and 72 for PHE and PYR, respectively.

  16. Pulsed field gel electrophoresis on frozen tumour tissue sections.

    PubMed Central

    Boultwood, J.; Kaklamanis, L.; Gatter, K. C.; Wainscoat, J. S.

    1992-01-01

    The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperature agarose. The tumour DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is yielded by this method to obtain a hybridisation signal with a single copy probe. Histological examination of adjacent tissue sections may also be carried out, permitting correlation between molecular analysis and tumour histology. Images PMID:1401187

  17. Extracellular matrix and cell surface as determinants of connective tissue differentiation.

    PubMed

    Solursh, M

    1989-09-01

    This paper reviews in vitro studies, largely from the author's laboratory, concerning the conditions that are permissive for the differentiation of limb bud mesenchymal cells into chondrocytes. In high-density cell culture, even in a defined medium, the same normal sequence of events that is found in vivo in developing cartilage is also observed. This system can be used to study heritable disorders in model systems such as in mutant mouse embryos. In addition, single mesenchymal cells can differentiate into hypertrophic chondrocytes in hydrated collagen gel or agarose cultures. A rounded cell shape promotes chondrogenesis, while a flattened cell shape promotes fibroblast differentiation. The actin cytoskeleton is shown to play a central role in regulating connective tissue cell differentiation. By use of such cell culture manipulations, it is now possible to grow large numbers of fibroblastic cells from human biopsy material for storage and to carry out experimental studies after re-expression of chondrogenesis in gel cultures. It is suggested that cytoskeletal-extracellular matrix interactions play a fundamental role in connective tissue differentiation. Matrix receptors might be developmentally regulated and modify epithelial effects on mesenchymal cells. In this way mesenchymal cells differentiate in a highly organized manner in spatial and temporal terms.

  18. Optimal processing for gel electrophoresis images: Applying Monte Carlo Tree Search in GelApp.

    PubMed

    Nguyen, Phi-Vu; Ghezal, Ali; Hsueh, Ya-Chih; Boudier, Thomas; Gan, Samuel Ken-En; Lee, Hwee Kuan

    2016-08-01

    In biomedical research, gel band size estimation in electrophoresis analysis is a routine process. To facilitate and automate this process, numerous software have been released, notably the GelApp mobile app. However, the band detection accuracy is limited due to a band detection algorithm that cannot adapt to the variations in input images. To address this, we used the Monte Carlo Tree Search with Upper Confidence Bound (MCTS-UCB) method to efficiently search for optimal image processing pipelines for the band detection task, thereby improving the segmentation algorithm. Incorporating this into GelApp, we report a significant enhancement of gel band detection accuracy by 55.9 ± 2.0% for protein polyacrylamide gels, and 35.9 ± 2.5% for DNA SYBR green agarose gels. This implementation is a proof-of-concept in demonstrating MCTS-UCB as a strategy to optimize general image segmentation. The improved version of GelApp-GelApp 2.0-is freely available on both Google Play Store (for Android platform), and Apple App Store (for iOS platform).

  19. Hybrid Gel Composed of Native Heart Matrix and Collagen Induces Cardiac Differentiation of Human Embryonic Stem Cells without Supplemental Growth Factors

    PubMed Central

    Duan, Yi; Liu, Zen; O'Neill, John; Wan, Leo Q.; Freytes, Donald O.; Vunjak-Novakovic, Gordana

    2011-01-01

    Our goal was to assess the ability of native heart extracellular matrix (ECM) to direct cardiac differentiation of human embryonic stem cells (hESCs) in vitro. In order to probe the effects of cardiac matrix on hESC differentiation, a series of hydrogels was prepared from decellularized ECM from porcine hearts by mixing ECM and collagen type I at varying ratios. Maturation of cardiac function in embryoid bodies formed from hESCs was documented in terms of spontaneous contractile behavior and the mRNA and protein expression of cardiac markers. Hydrogel with high ECM content (75% ECM, 25% collagen, no supplemental soluble factors) increased the fraction of cells expressing cardiac marker troponin T, when compared with either hydrogel with low ECM content (25% ECM, 75% collagen, no supplemental soluble factors) or collagen hydrogel (100% collagen, with supplemental soluble factors). Furthermore, cardiac maturation was promoted in high-ECM content hydrogels, as evidenced by the striation patterns of cardiac troponin I and by upregulation of Cx43 gene. Consistently, high-ECM content hydrogels improved the contractile function of cardiac cells, as evidenced by increased numbers of contracting cells and increased contraction amplitudes. The ability of native ECM hydrogel to induce cardiac differentiation of hESCs without the addition of soluble factors makes it an attractive biomaterial system for basic studies of cardiac development and potentially for the delivery of therapeutic cells into the heart. PMID:21744185

  20. Achondrogenesis type IB (Fraccaro): study of collagen in the tissue and in chondrocytes cultured in agarose.

    PubMed

    Freisinger, P; Stanescu, V; Jacob, B; Cohen-Solal, L; Maroteaux, P; Bonaventure, J

    1994-02-15

    A lethal chondrodysplasia characterized by extreme micromelia was diagnosed by ultrasound examination in two sibs whose nonconsanguineous parents were healthy. Radiographic and histopathologic data indicated that the two foetuses (18 and 21 weeks old) had achondrogenesis type IB (Fraccaro). Quantitation of total collagen extractable from dried cartilage samples demonstrated a 50% decrease when compared to an age-related control. This decrease was essentially related to type II collagen. Nevertheless, the alpha chains and the CB peptides of type II collagen had a normal electrophoretic mobility. A significant amount of collagen type I was also detected. The electrophoretic pattern of collagens type IX and XI did not differ significantly from control sample. The extracellular matrix elaborated by patient chondrocytes cultured in agarose for 10-12 days, contained less collagen type II than normal cells. Labelling with 14C-proline of cultured cells showed the presence of procollagen and type II collagen chains with a normal electrophoretic mobility, but an alpha 2(I) chain was detectable in the patient material, indicating the presence of collagen type I which supported the tissue findings. The significance of the type II collagen reduction in the patient's cartilage is unclear but it is unlikely to be the primary defect in achondrogenesis type I.

  1. Ni{sub 0.5}Zn{sub 0.5}Fe{sub 2}O{sub 4} nanoparticles dispersed in a SiO{sub 2} matrix synthesized by sol-gel processing

    SciTech Connect

    Pozo Lopez, G.; Condo, A.M.; Urreta, S.E.; Silvetti, S.P.; Aguirre, M. del C.

    2012-12-15

    (Ni{sub 0.5}Zn{sub 0.5}Fe{sub 2}O{sub 4})x/(SiO{sub 2})(100 - x) (x = 5, 20 and 50 wt.%) nanocomposites are synthesized by a sol-gel method using tetraethylorthosilicate (TEOS) and metallic nitrates as precursors, and by further annealing the powders for 1 h at 1273 K. X-ray diffraction (XRD), transmission electron microscopy (TEM), room temperature vibrating sample magnetometry (VSM) and SQUID measurements are employed for structural, morphological and magnetic sample characterization. For all the concentrations analyzed, the powder nanocomposites actually consist of spinel NiZn ferrite nanoparticles, dispersed in an amorphous silica matrix. TEM studies reveal different particle size distributions and particle morphologies for the three ferrite contents. The 20 wt.%-NiZn ferrite samples consist of nearly spherical nanoparticles, of about 8 nm, mainly superparamagnetic, well-dispersed in the amorphous silica matrix, while the 5 wt.%-NiZn ferrite samples exhibit a bimodal particle size distribution (5 and 30 nm) of single-domain nanoparticles embedded in the silica. In the 50 wt.%-NiZn ferrite samples, two particle families are observed: small round superparamagnetic nanoparticles of about 8 nm embedded in the amorphous silica matrix and large, non-spherical, ferrimagnetic ones, forming agglomerates outside the matrix. In all the synthesized samples, thickness fringes are observed inside some of the ferrite nanoparticles in dark field images. This contrast is explained using the theory of electron diffraction in a weak beam dark field (WBDF) condition and considering spherical ferrite nanoparticles. A large range of tailored magnetic properties varying the fraction, dispersion and mean size of the ferrimagnetic NiZn ferrite particles is obtained. Room temperature saturation magnetization values are found in the range 3.0-30.4 Am{sup 2}/kg for the different concentration samples. Coercivity values, between 1.9 and 7.6 mT, are more than 50% higher than those measured

  2. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

  3. Copolymers For Capillary Gel Electrophoresis

    SciTech Connect

    Liu, Changsheng; Li, Qingbo

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  4. Time Controlled Protein Release from Layer-by-Layer Assembled Multilayer Functionalized Agarose Hydrogels

    PubMed Central

    Mehrotra, Sumit; Lynam, Daniel; Maloney, Ryan; Pawelec, Kendell M.; Tuszynski, Mark H.; Lee, Ilsoon

    2009-01-01

    Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injury. Experimentally generated patterns of axon growth are typically disorganized and randomly oriented. Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogel, and containing genetically engineered cells that secrete brain-derived neurotrophic factor (BDNF). However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and alternative strategies are needed to provide sustained release of BDNF from templated agarose scaffolds. Existing methods of loading the drug or protein into hydrogels cannot provide sustained release from templated agarose hydrogels. Alternatively, here it is shown that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-by-layer (LbL) thin films, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. Lysozyme, a protein similar in size and isoelectric point to BDNF, is released from the multilayers on the agarose and is biologically active during the earlier time points, with decreasing activity at later time points. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein is loaded separately from the agarose hydrogel fabrication process. PMID:20200599

  5. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

    PubMed

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

    Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond

  6. Living bacteria in silica gels

    NASA Astrophysics Data System (ADS)

    Nassif, Nadine; Bouvet, Odile; Noelle Rager, Marie; Roux, Cécile; Coradin, Thibaud; Livage, Jacques

    2002-09-01

    The encapsulation of enzymes within silica gels has been extensively studied during the past decade for the design of biosensors and bioreactors. Yeast spores and bacteria have also been recently immobilized within silica gels where they retain their enzymatic activity, but the problem of the long-term viability of whole cells in an inorganic matrix has never been fully addressed. It is a real challenge for the development of sol-gel processes. Generic tests have been performed to check the viability of Escherichia coli bacteria in silica gels. Surprisingly, more bacteria remain culturable in the gel than in an aqueous suspension. The metabolic activity of the bacteria towards glycolysis decreases slowly, but half of the bacteria are still viable after one month. When confined within a mineral environment, bacteria do not form colonies. The exchange of chemical signals between isolated bacteria rather than aggregates can then be studied, a point that could be very important for 'quorum sensing'.

  7. Identification and comparative proteomic study of quail and duck egg white protein using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis.

    PubMed

    Hu, S; Qiu, N; Liu, Y; Zhao, H; Gao, D; Song, R; Ma, M

    2016-05-01

    A proteomic study of egg white proteins from 2 major poultry species, namely quail (Coturnix coturnix) and duck (Anas platyrhynchos), was performed with comparison to those of chicken (Gallus gallus) through 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis. By using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), 29 protein spots representing 10 different kinds of proteins as well as 17 protein spots designating 9 proteins were successfully identified in quail and duck egg white, respectively. This report suggested a closer relationship between quail and chicken egg white proteome patterns, whereas the duck egg white protein distribution on the 2-DE map was more distinct. In duck egg white, some well-known major proteins, such as ovomucoid, clusterin, extracellular fatty acid-binding protein precursor (ex-FABP), and prostaglandin D2 synthase (PG D2 synthase), were not detected, while two major protein spots identified as "deleted in malignant brain tumors 1" protein (DMBT1) and vitellogenin-2 were found specific to duck in the corresponding range on the 2-DE gel map. These interspecies diversities may be associated with the egg white protein functions in cell defense or regulating/supporting the embryonic development to adapt to the inhabiting environment or reproduction demand during long-term evolution. The findings of this work will give insight into the advantages involved in the application on egg white proteins from various egg sources, which may present novel beneficial properties in the food industry or related to human health.

  8. Influence of calcination temperature on structural and magnetic properties of nanocomposites formed by Co-ferrite dispersed in sol-gel silica matrix using tetrakis(2-hydroxyethyl) orthosilicate as precursor

    PubMed Central

    2011-01-01

    Effects of calcination temperatures varying from 400 to 1000°C on structural and magnetic properties of nanocomposites formed by Co-ferrite dispersed in the sol-gel silica matrix using tetrakis(2-hydroxyethyl) orthosilicate (THEOS) as water-soluble silica precursor have been investigated. Studies carried out using XRD, FT-IR, TEM, STA (TG-DTG-DTA) and VSM techniques. Results indicated that magnetic properties of samples such as superparamagnetism and ferromagnetism showed great dependence on the variation of the crystallinity and particle size caused by the calcination temperature. The crystallization, saturation magnetization Ms and remenant magnetization Mr increased as the calcination temperature increased. But the variation of coercivity Hc was not in accordance with that of Ms and Mr, indicating that Hc is not determined only by the crystallinity and size of CoFe2O4 nanoparticles. TEM images showed spherical nanoparticles dispersed in the silica network with sizes of 10-30 nm. Results showed that the well-established silica network provided nucleation locations for CoFe2O4 nanoparticles to confinement the coarsening and aggregation of nanoparticles. THEOS as silica matrix network provides an ideal nucleation environment to disperse CoFe2O4 nanoparticles and thus to confine them to aggregate and coarsen. By using THEOS as water-soluble silica precursor over the currently used TEOS and TMOS, the organic solvents are not needed owing to the complete solubility of THEOS in water. Synthesized nanocomposites with adjustable particle sizes and controllable magnetic properties make the applicability of Co-ferrite even more versatile. PMID:21486494

  9. Highly sensitive analysis of nucleic acids using capillary gel electrophoresis with ultraviolet detection based on the combination of matrix field-amplified and head-column field-amplified stacking injection.

    PubMed

    Lian, Dong-Sheng; Zhao, Shu-Jin

    2015-01-26

    To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13 ng/ml (signal/noise=3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area (n=3) were 0.03-1.15 and 0.72-6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise=3), The relative standard deviations for migration time and peak area (n=6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.

  10. Dynamics of DNA molecules under gel electrophoresis

    SciTech Connect

    Kotaka, Tadao, Adachi, Shiro; Shikata, Toshiyuki

    1993-12-31

    Electrophoretic mobilities {mu} of double stranded linear DNAs were examined in agarose gels subjected to a biased sinusoidal field (BSF) that utilizes a sinusoidal field of strength E{sub s} and frequency f superposed on a steady bias field of strength E{sub b}. Under BSF with E{sub s} {much_gt} E{sub b}. DNA fragments with the size M > 20 kbp exhibited peculiar behavior which the authors called a pin down phenomenon in that the {mu} shows a minimum {mu}{sub p} at a particular f{sub p} (pin down frequency) specific to M, C{sub gel} and the field strengths. The dynamics of DNA molecules under such pin-down conditions were examined by direct observation via fluorescence microscopy as well as dynamic electric birefringence.

  11. Elastic and macroporous agarose-gelatin cryogels with isotropic and anisotropic porosity for tissue engineering.

    PubMed

    Tripathi, Anuj; Kathuria, Neeraj; Kumar, Ashok

    2009-09-01

    The focus of this work was to design a macroporous scaffold with controlled porosity in isotropic and anisotropic manner for tissue-engineering applications. Agarose-gelatin scaffolds were synthesized by cryogelation method, in which agarose was used to improve the mechanical characteristics and gelatin-provided amiable property of elasticity, cell adhesion, and cell proliferation in the scaffold. Agarose-gelatin (8%) cryogels synthesized in two different solvent systems (i.e., water and 0.1% acetic acid) at subzero temperature (-12 degrees C) showed well-interconnected porous structure. The agarose-gelatin cryogel synthesized in water solvent system (WSS) showed gradient porosity with an average pore diameter of a monolith (four sections from bottom to top; height 5 mm and diameter 13 mm each) ranging from 76 to 187 microm. The monolith of agarose-gelatin synthesized in 0.1% acetic acid solvent system (0.1% ASS) did not show any remarkable difference in average pore diameter of a monolith to their whole column length as revealed by scanning electron microscopy (SEM). These cryogels swelled up to approximately 90% of their capacity within 1 min. The aggregate tensile modulus showed good elasticity of the cryogels, in which agarose-gelatin synthesized in WSS showed higher tensile modulus, that is, 380.23 +/- 63.97 kPa in comparison with agarose-gelatin synthesized in 0.1% ASS, i.e., 278.08 +/- 94.08 kPa. The unconfined fatigue observation with varying strain (10-40%) and varying frequencies (2 and 5 Hz) showed no deformation of cryogels. The fibroblast (Cos-7) cell line seeded on the scaffold displayed good cell attachment in both types of cryogels and MTT assay showed good cell compatibility and favorable conditions for cell proliferation. These results indicate that agarose-gelatin cryogels can be a promising material of choice for tissue-engineering applications.

  12. A new material for tissue engineered vagina reconstruction: Acellular porcine vagina matrix.

    PubMed

    Zhang, Jing-Kun; Du, Run-Xuan; Zhang, Lin; Li, Ya-Nan; Zhang, Ming-le; Zhao, Shuo; Huang, Xiang-Hua; Xu, Yan-Fang

    2017-03-10

    Acellular matrix materials have been widely used to repair various tissues and organs. According to the plastic principle, when a part of the body is lost, it should be replaced with a similar material. Therefore, the use of a homologous organ-specific acellular vaginal tissue in vagina reconstruction repair surgery may show good results. However, the acellular vagina matrix (AVM) form large vertebrates is difficult to isolate. In this study, we described a multi-step method to prepare porcine AVM and evaluated the efficacy of acellularization. We also investigated the biomechanical properties, biological activity elements and biocompatibility of the porcine AVM. We then used this material to reconstruct a rat vagina and performed further morphologic and functional analyses. Small intestinal submucosa (SIS), which is a commonly used acellular matrix material, was used in a control group. Histological examination, DNA content analysis and agarose gel electrophoresis revealed that the decellularization procedure was effective. The AVM had acceptable biomechanical properties and sufficient growth factor production (VEGF, FGF, TGF-β1 and PDGF-BB) compared with that of the SIS. Subcutaneous transplantation in rats showed that the AVM had good biocompatibility. The tissue-engineered vagina using the AVM more resembled normal-appearing tissue than did that using SIS following morphologic and functional analyses. The AVM has great potential for application in vaginal reconstructive surgery. This article is protected by copyright. All rights reserved.

  13. Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

    PubMed

    Fuchigami, Takashi; Misumi, Shogo; Takamune, Nobutoki; Takahashi, Ichiro; Takama, Michiho; Shoji, Shozo

    2002-05-10

    HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.

  14. The Effect of Autologous Platelet-Rich Gel on the Dynamic Changes of the Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-2 Expression in the Diabetic Chronic Refractory Cutaneous Ulcers.

    PubMed

    Li, Lan; Chen, Dawei; Wang, Chun; Liu, Guanjian; Ran, Xingwu

    2015-01-01

    Aim. To investigate the dynamic changes on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in the diabetic chronic refractory cutaneous ulcers after the autologous platelet-rich gel (APG) treatment. Methods. The study was developed at the Diabetic Foot Care Centre, West China Hospital. The granulation tissues from the target wounds were taken before and within 15 days after APG application. The expression of MMP-2 and TIMP-2 as well as transforming growth factor-β1 (TGF-β1) in the granulation tissue was detected by q TR-PCR and IHC. The relationship between the expression level of MMP-2 and TIMP-2 and their ratio and that of TGF-β1 was analyzed. Results. The expression of MMP-2 (P < 0.05) was suppressed, and the expression of TIMP-2 (P < 0.05) was promoted, while the ratio of MMP-2/TIMP-2 (P < 0.05) was decreased after APG treatments. The expression of TGF-β1 had negative correlation with the ratio of MMP-2/TIMP-2 (P < 0.05) and positive correlation with the expression of TIMP-2 (P < 0.05). Conclusions. APG treatment may suppress the expression of MMP-2, promoting that of the TIMP-2 in the diabetic chronic refractory cutaneous wounds. TGF-β1 may be related to these effects.

  15. Electrokinetics of nanoparticle gel-electrophoresis.

    PubMed

    Hill, Reghan J

    2016-09-28

    -electrophoresis experiments have recently been applied. To demonstrate its practical application, the model is applied to (pH charge regulating) carboxylated polystyrene nanospheres in low-density passivated agarose gels (weak steric effects). This furnishes a new theoretical interpretation of literature data for which a finite diffuse-layer-thickness, pH-charge regulation, high charge, and relaxation effects dominate over the steric influences.

  16. Single nucleus versus single-cell gel electrophoresis: kinetics of DNA track formation.

    PubMed

    Afanasieva, Katerina; Chopei, Marianna; Sivolob, Andrei

    2015-04-01

    Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single-nucleus gel electrophoresis may be a way for the comet assay standardization.

  17. Fabricating neuromast-inspired gel structures for membrane-based hair cell sensing

    NASA Astrophysics Data System (ADS)

    Tamaddoni, Nima J.; Stephens, Christopher P.; Sarles, S. A.

    2012-04-01

    Recent research has shown that a new class of mechanical sensor, assembled from biomolecules and which features an artificial cell membrane as the sensing element, can be used to mimic basic hair cell mechanotransduction in vertebrates. The work presented in this paper is motivated by the need to increase sensor performance and stability by refining the methods used to fabricate and connect lipid-encapsulated hydrogels. Inspired by superficial neuromasts found on fish, three hydrogel materials are compared for their ability to be readily shaped into neuromast-inspired geometries and enable lipid bilayer formation using self-assembly at an oil/water interface. Agarose, polyethylene glycol (PEG, 6kg/mole), and hydroxyethyl methacrylate (HEMA) gel materials are compared. The results of this initial study determined that UV-curable gel materials such as PEG and HEMA enable more accurate shaping of the gel-needed for developing a sensor that uses a gel material both for mechanical support and membrane formation-compared to agarose. However, the lower hydrophobicity of agarose and PEG materials provide a more fluid, water-like environment for membrane formation-unlike HEMA. In working toward a neuromast-inspired design, a final experiment demonstrates that a bilayer can also be formed directly between two lipid-covered PEG surfaces. These initial results suggest that candidate gel materials with a low hydrophobicity, high fluidity, and a low modulus can be used to provide membrane support.

  18. Aerosol gels

    NASA Technical Reports Server (NTRS)

    Sorensen, Christopher M. (Inventor); Chakrabarti, Amitabha (Inventor); Dhaubhadel, Rajan (Inventor); Gerving, Corey (Inventor)

    2010-01-01

    An improved process for the production of ultralow density, high specific surface area gel products is provided which comprises providing, in an enclosed chamber, a mixture made up of small particles of material suspended in gas; the particles are then caused to aggregate in the chamber to form ramified fractal aggregate gels. The particles should have a radius (a) of up to about 50 nm and the aerosol should have a volume fraction (f.sub.v) of at least 10.sup.-4. In preferred practice, the mixture is created by a spark-induced explosion of a precursor material (e.g., a hydrocarbon) and oxygen within the chamber. New compositions of matter are disclosed having densities below 3.0 mg/cc.

  19. Gel mesh as ``brake'' to slow down DNA translocation through solid-state nanopores

    NASA Astrophysics Data System (ADS)

    Tang, Zhipeng; Liang, Zexi; Lu, Bo; Li, Ji; Hu, Rui; Zhao, Qing; Yu, Dapeng

    2015-07-01

    Agarose gel is introduced onto the cis side of silicon nitride nanopores by a simple and low-cost method to slow down the speed of DNA translocation. DNA translocation speed is slowed by roughly an order of magnitude without losing signal to noise ratio for different DNA lengths and applied voltages in gel-meshed nanopores. The existence of the gel moves the center-of-mass position of the DNA conformation further from the nanopore center, contributing to the observed slowing of translocation speed. A reduced velocity fluctuation is also noted, which is beneficial for further applications of gel-meshed nanopores. The reptation model is considered in simulation and agrees well with the experimental results.Agarose gel is introduced onto the cis side of silicon nitride nanopores by a simple and low-cost method to slow down the speed of DNA translocation. DNA translocation speed is slowed by roughly an order of magnitude without losing signal to noise ratio for different DNA lengths and applied voltages in gel-meshed nanopores. The existence of the gel moves the center-of-mass position of the DNA conformation further from the nanopore center, contributing to the observed slowing of translocation speed. A reduced velocity fluctuation is also noted, which is beneficial for further applications of gel-meshed nanopores. The reptation model is considered in simulation and agrees well with the experimental results. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03084f

  20. Direct noninvasive measurement and numerical modeling of depth-dependent strains in layered agarose constructs.

    PubMed

    Griebel, A J; Khoshgoftar, M; Novak, T; van Donkelaar, C C; Neu, C P

    2014-06-27

    Biomechanical factors play an important role in the growth, regulation, and maintenance of engineered biomaterials and tissues. While physical factors (e.g. applied mechanical strain) can accelerate regeneration, and knowledge of tissue properties often guide the design of custom materials with tailored functionality, the distribution of mechanical quantities (e.g. strain) throughout native and repair tissues is largely unknown. Here, we directly quantify distributions of strain using noninvasive magnetic resonance imaging (MRI) throughout layered agarose constructs, a model system for articular cartilage regeneration. Bulk mechanical testing, giving both instantaneous and equilibrium moduli, was incapable of differentiating between the layered constructs with defined amounts of 2% and 4% agarose. In contrast, MRI revealed complex distributions of strain, with strain transfer to softer (2%) agarose regions, resulting in amplified magnitudes. Comparative studies using finite element simulations and mixture (biphasic) theory confirmed strain distributions in the layered agarose. The results indicate that strain transfer to soft regions is possible in vivo as the biomaterial and tissue changes during regeneration and maturity. It is also possible to modulate locally the strain field that is applied to construct-embedded cells (e.g. chondrocytes) using stratified agarose constructs.

  1. Network generation enhances interpretation of proteomics data sets by a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

    PubMed

    Wang, Xijun; Zhang, Aihua; Sun, Hui; Wu, Gelin; Sun, Wenjun; Yan, Guangli

    2012-10-21

    Recent advances in proteomic technologies have enabled us to create detailed protein-protein interaction maps in diseases. As the size of the interaction dataset increases, powerful computational methods are required in order to effectively interpret network models from large scale interactome data. In this study, we carried out comparative proteomics to construct and identify the proteins networks associated with hepatic injury (HI) which are largely unknown, as a case study. All proteins expressed were separated and identified by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Protein-interacting networks and pathways were mapped using STRING analysis program. We have performed for the first time a comprehensive profiling of changes in protein expression of HI rats, to uncover the networks altered by treated with CCl(4). Identification of fifteen spots (seven over-expressed and eight under-expressed) were established by MALDI-TOF/TOF MS. These proteins were subjected to functional pathway analysis using STRING software for better understanding of the biological context of the identified proteins. It suggested that modulation of multiple vital physiological pathways including DNA repair process, cell apoptosis, oxidation reduction, signal transduction, metabolic process, intracellular signaling cascade, regulation of biological processes, cell communication, regulation of cellular process, and molecular transport. In summary, the present study provides the first protein-interacting network maps and novel insights into the biological responses and potential pathways of HI. The generation of protein interaction networks clearly enhances the interpretation of proteomic data, particularly in respect of understanding molecular mechanisms of panel protein biomarkers.

  2. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    SciTech Connect

    Kovacs, E.; Langemann, H. )

    1990-11-01

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of (3H)thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.

  3. Preparation of DNA and protein micro arrays on glass slides coated with an agarose film

    PubMed Central

    Afanassiev, Victor; Hanemann, Vera; Wölfl, Stefan

    2000-01-01

    A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents. PMID:10871389

  4. Quantitative determination of glycine in aqueous solution using glutamate dehydrogenase-immobilized glyoxal agarose beads.

    PubMed

    Keskin, Semra Yilmazer; Keskin, Can Serkan

    2014-01-01

    In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD(+) in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1-10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).

  5. Brain derived neurotrophic factor release from layer-by-layer coated agarose nerve guidance scaffolds.

    PubMed

    Lynam, Daniel A; Shahriari, Dena; Wolf, Kayla J; Angart, Phillip A; Koffler, Jacob; Tuszynski, Mark H; Chan, Christina; Walton, Patrick; Sakamoto, Jeffrey

    2015-05-01

    Agarose nerve guidance scaffolds (NGS) seeded with cells expressing brain derived neurotrophic factor (BDNF) have demonstrated robust nerve regeneration in the rat central nervous system. The purpose of this work was to explore whether agarose NGS coated with hydrogen-bonded layer-by-layer (HLbL) could provide an acellular method of delivering prolonged and consistent dosages of active BDNF. Our results show that HLbL-coated agarose NGS could release BDNF over 10days in consistent dosages averaging 80.5±12.5(SD)ng/mL. Moreover, the BDNF released from HLbL was confirmed active by in vitro cell proliferation assays. To our knowledge, this is the first report demonstrating that HLbL assembled onto a hydrogel can provide consistent, prolonged release of active BDNF in clinically relevant dosages.

  6. Passivated gel electrophoresis of charged nanospheres by light-scattering video tracking.

    PubMed

    Zhu, Xiaoming; Mason, Thomas G

    2014-08-15

    Gel electrophoresis (gel-EP) has been used for decades to separate charged biopolymers, such as DNA, RNA, and proteins, yet propagation of other charged colloidal objects, such as nanoparticles, during gel-EP has been studied comparatively little. Simply introducing anionic nanoparticles, such as sulfate-stabilized polystyrene nanospheres, in standard large-pore agarose gels commonly used for biomolecules does not automatically ensure propagation or size-separation because attractive interactions can exist between the gel and the nanoparticles. Whereas altering the surfaces of the nanoparticles is a possible solution, here, by contrast, we show that treating a common type I-A low-electroendoosmosis agarose gel with a passivation agent, such as poly-(ethyleneglycol), enables charged nanoparticles to propagate through large-pore passivated gels in a highly reproducible manner. Moreover, by taking advantage of the significant optical scattering from the nanoparticles, which is not easily measurable for biopolymers, relative to scattering from the gel, we perform real-time, light-scattering, video-tracking gel-EP. Continuous optical measurements of the propagation of bands of uniformly sized nanospheres in passivated gels provides the propagation distance, L, and velocity, v, as a function of time for different sphere radii, electric field strengths, gel concentrations, and passivation agent concentrations. The steady-state particle velocities vary linearly with applied electric field strength, E, for small E, but these velocities become non-linear for larger E, suggesting that strongly driven nanoparticles can become elastically trapped in the smaller pores of the gel, which act like blind holes, in a manner that thermal fluctuations cannot overcome. Based on this assumption, we introduce a simple model that fits the measured v(E) in both linear and non-linear regimes over a relevant range of applied voltages.

  7. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing.

    PubMed

    Dvorak, H F; Harvey, V S; Estrella, P; Brown, L F; McDonagh, J; Dvorak, A M

    1987-12-01

    Fibrin deposition is a consistent early event in solid tumors and healing wounds and precedes new blood vessel ingrowth in both. We now demonstrate that fibrin gels of themselves induce an angiogenic response in the absence of tumor cells or platelets. Angiogenesis was enhanced when certain chemoattractants or mitogens were included in the fibrin gel. Newly devised, inert plastic chambers with one porous surface were filled with varying contents and were implanted in the subcutaneous space of guinea pigs. Chambers filled with cross-linked homologous fibrin or plasma induced an angiogenic response within 4 days. Vessels entered chambers through the surface pores and flared out radially; angiogenesis was quantitated by point counting. Vessels were functional and matured along a gradient that proceeded from distal (least mature) to proximal. The intensity of the angiogenic response was enhanced when zymosan activated serum, an N-formylmethionine tripeptide, or platelet-derived growth factor was included in the fibrin matrix. Prior aldehyde fixation or boiling of fibrin-filled chambers inhibited angiogenesis, as did high concentrations of hyaluronic acid. Chambers filled with type I collagen or agarose did not induce new blood vessel formation, but addition of collagen did not reduce fibrin's capacity to initiate angiogenesis. The novel assay introduced here offers several advantages that should facilitate the study of angiogenesis. These include reproducibility, low background, objective and quantitative scoring, and the capacity to evaluate native molecules in animals of several species. Taken together, our findings strongly implicate fibrin or related proteins in the pathogenesis of angiogenesis and offer a new approach for elucidating the underlying molecular mechanisms.

  8. Drying techniques for the visualisation of agarose-based chromatography media by scanning electron microscopy.

    PubMed

    Nweke, Mauryn C; Turmaine, Mark; McCartney, R Graham; Bracewell, Daniel G

    2017-03-01

    The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub-micron level. Achieving suitable drying conditions is especially important with agarose-based chromatography resins, as over-drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under-drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect™/dw ≈85 µm and Capto™ Adhere/dw ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose-based chromatography media.

  9. Can You Solve the Crime? Using Agarose Electrophoresis To Identify an Unknown Colored Protein.

    ERIC Educational Resources Information Center

    Wiltfong, Cynthia L.; Chester, Emily; Albertin, Faith; Smith, Julia; Hall, Judith C.; Arth, Emily C.; Martin, Stephanie

    2003-01-01

    Describes a lab that introduces agarose electrophoresis techniques and basic information on proteins to middle school and high school students. Insists that, built around a scenario in which students must solve a crime, the lab has real-world applications that should spark student interest. (KHR)

  10. Agarose-Based Substrate Modification Technique for Chemical and Physical Guiding of Neurons In Vitro.

    PubMed

    Krumpholz, Katharina; Rogal, Julia; El Hasni, Akram; Schnakenberg, Uwe; Bräunig, Peter; Bui-Göbbels, Katrin

    2015-08-26

    A new low cost and highly reproducible technique is presented that provides patterned cell culture substrates. These allow for selective positioning of cells and a chemically and mechanically directed guiding of their extensions. The patterned substrates consist of structured agarose hydrogels molded from reusable silicon micro templates. These templates consist of pins arranged equidistantly in squares, connected by bars, which mold corresponding wells and channels in the nonadhesive agarose hydrogel. Subsequent slice production with a standard vibratome, comprising the described template pattern, completes substrate production. Invertebrate neurons of locusts and pond snails are used for this application as they offer the advantage over vertebrate cells as being very large and suitable for cultivation in low cell density. Their neurons adhere to and grow only on the adhesive areas not covered by the agarose. Agarose slices of 50 μm thickness placed on glass, polystyrene, or MEA surfaces position and immobilize the neurons in the wells, and the channels guide their neurite outgrowth toward neighboring wells. In addition to the application with invertebrate neurons, the technique may also provide the potential for the application of a wide range of cell types. Long-term objective is the achievement of isolated low-density neuronal networks on MEAs or different culture substrates for various network analysis applications.

  11. Porous Agarose-Based Semi-IPN Hydrogels: Characterization and Cell Affinity Studies.

    PubMed

    Vardar, E; Vert, Michel; Coudane, Jean; Hasirci, V; Hasirci, N

    2012-01-01

    Hydrogels are frequently considered for medical applications due to the ease of preparation in different forms and high water content that makes them comparable to natural tissues. However, these general properties are not sufficient to make any hydrogel suitable for cell attachment and growth which are necessary for their use in tissue regeneration. Besides, the high water content makes the hydrogels mechanically weak. The formation of semi-interpenetrating networks (semi-IPNs) can be used in attempts to enhance physical, mechanical and thermal properties. In this study, semi-IPNs of agarose were prepared with chitosan and alginate, two polyelectrolytes that are positively and negatively charged under physiological conditions, respectively. Zeta potential was used to confirm the formation of charged hydrogels. All hydrogels had ultimate compression strengths in the range of 91-210 Pa where the value for pure agarose was about 103 Pa. Chitosan increased the compressive strength about two folds whereas the alginate had opposite effects. The amount of strongly bound water present in the hydrogels were estimated from TGA and DSC analysis and the highest value was found for alginate-agarose hydrogels as about 15%. The attachment and the migration of L929 fibroblasts were monitored in vitro using the MTS assay and confocal microscopy. The highest cell proliferation and penetration were observed for positively charged chitosan-agarose semi-IPN hydrogels.

  12. Modification of agarose: 6-aminoagarose mediated syntheses of fluorogenic pyridine carboxylic acid amides.

    PubMed

    Kondaveeti, Stalin; Mehta, Gaurav K; Siddhanta, A K

    2014-06-15

    A facile 6-aminoagarose (AA) mediated synthesis of new fluorogenic amides of agarose with nicotinic (AA-NA) and picolinic acids (AA-PA) employing carbodiimide chemistry have been described. 6-Amino agarose (AA) was synthesized in a facile Mitsunobu-inspired microwave mediated method involving the reaction of agarose with phthalimide in presence of diisopropyl azodicarboxylate and triphenylphosphene (DIAD/TPP) followed by hydrazinolysis. All compounds were characterized by GPC, UV spectrophotometry, fluorescence spectroscopy, FT-IR, (1)H and (13)C NMR spectra. The fluorescence emissions (λmax 430 and 412 nm) of 1 × 10(-3)M solutions of AA-NA and AA-PA in water were significantly higher (ca. 82% and ca. 90%) than those of the molar equivalents (0.2mg) of NA and PA present in the 1 × 10(-3)M solutions of the amides, respectively. These fluorogenic pyridine carboxylic acid amides of agarose may find applications as sensors in biomedical and pharmaceutical industries.

  13. Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.

    PubMed

    Perła, Joanna; Undas, Anetta; Twardowski, Tomasz; Jakubowski, Hieronim

    2004-08-05

    Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

  14. A templated agarose scaffold for axon guidance in the central and peripheral nervous system

    NASA Astrophysics Data System (ADS)

    Gros, Thomas Richard

    This thesis examined the hypothesis that axonal guidance could be improved in the central and peripheral nervous systems using a highly linearized templated agarose scaffold. In the present study we examined whether a templated agarose scaffold improved axon retention across a large central nervous system (CNS) lesion and how cellular and axonal orientation was affected within the scaffold channels. The "physical" guidance from the scaffold was applied to an existing CNS "chemical" guidance strategy, shown to promote axons beyond the lesion site, to enhance the number of crossing axons in larger, disorganized, lesions. Specifically, there was the greatest number of long-tract sensory axons reaching the distal aspect of the lesion when the templated agarose scaffold was combined with a neurotrophic source of NT-3 beyond the lesion site and a conditioning lesion, to enhance chemical axon guidance and the intrinsic growth state of axons, respectively. When comparing the scaffold implant to a cell suspension grafts, we found a higher retention of long-tract ascending (sensory) axons and descending (motor) axons crossing large lesions (2mm). The enhanced axon retention may be attributed to the finding that cellular orientation within the scaffold channels is highly linear, thus promoting a less tortuous environment for axon orientation and bridging. Although an enhanced number of axons were able to cross the lesion, the axons did not repenetrate the host tissue due to a reactive cell layer, present only in scaffold the implant groups. Additionally, a peripheral nerve conduit, with the agarose scaffold as the core, displayed biocompatiablility and supported axon growth and vasculature beyond the clinically applicable distance of 4mm. Thus, the templated agarose scaffold enhances axon retention and guidance within CNS injury sites and has potential applications to the PNS.

  15. Analysis of DAPI and SYBR Green I as Alternatives to Ethidium Bromide for Nucleic Acid Staining in Agarose Gel Electrophoresis

    NASA Astrophysics Data System (ADS)

    Bourzac, Kevin M.; Lavine, Lori J.; Rice, Margaret S.

    2003-11-01

    DNA electrophoresis and staining is a common procedure in biochemistry laboratories, but the use of ethidium bromide (EB) for DNA detection is worrisome as EB is a mutagen and probable carcinogen. Five alternative stains were evaluated for DNA detection, safety, cost, and ease of use: BlueView, methylene blue, Carolina Blu, DAPI (4',6-diamidino-2-phenylindole dihydrochloride:hydrate), and SYBR Green I. BlueView, Carolina Blu, and methylene blue are not sensitive enough to detect the microgram amounts of DNA used in many procedures. However, DAPI and SYBR Green I are good staining alternatives to ethidium bromide in that they have similar sensitivity and are both easy to use. SYBR Green I is more expensive than EB or DAPI; however, the limited safety data suggest that SYBR Green I is the safest stain.

  16. Agarose Gel Electrophoresis System in the Classroom: Detection of DNA Strand Breaks through the Alteration of Plasmid Topology

    ERIC Educational Resources Information Center

    De Mattos, J. C. P.; Dantas, F. J. S.; Caldeira-de-Araujo, A.; Moraes, M. O.

    2004-01-01

    Good quality scientific teaching depends on the ability of researchers to translate laboratory experiments into high school and undergraduate classes, bridging the advanced and basic science with common knowledge. A fast-growing field in biomedical sciences is oxidative stress, which has been associated to several diseases, including cancer and…

  17. Extracellular production of a novel endo-β-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose.

    PubMed

    Hsu, Pang-Hung; Wei, Chien-Han; Lu, Wen-Jung; Shen, Fen; Pan, Chorng-Liang; Lin, Hong-Ting Victor

    2015-03-11

    The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.

  18. Extracellular Production of a Novel Endo-β-Agarase AgaA from Pseudomonas vesicularis MA103 that Cleaves Agarose into Neoagarotetraose and Neoagarohexaose

    PubMed Central

    Hsu, Pang-Hung; Wei, Chien-Han; Lu, Wen-Jung; Shen, Fen; Pan, Chorng-Liang; Lin, Hong-Ting Victor

    2015-01-01

    The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. PMID:25768342

  19. Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

    PubMed Central

    Bonza, Cristina; Carnelli, Antonella; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope. PMID:9490776

  20. A facile one-pot synthesis of a fluorescent agarose-O-naphthylacetyl adduct with slow release properties.

    PubMed

    Kondaveeti, Stalin; Chejara, Dharmesh R; Siddhanta, A K

    2013-10-15

    A microwave assisted facile synthesis of a fluorescent 6-O-naphthylacetyl agarose (NA-agarose) employing carbodiimide chemistry (dicyclohexylcarbodiimide/4-dimethylaminopyridine) has been described. NA-agarose was characterized by TGA, GPC, UV spectrophotometry, fluorescence spectroscopy, FT-IR, (1)H and (13)C NMR spectra, exhibiting that in NA-agarose the naphthylacetyl group was attached to the backbone of the agarose polymer. The hydrolysis of NA-agarose in heterogeneous aqueous phase showed that the 1-naphthyl acetic acid (NAA), a plant growth regulator, got released in a controlled manner, the release rate being dependent on the hydrophilicity of the polymer adduct as well as on pH and temperature. The fluorescence emission (λmax 332 nm) of NA-agarose (1×10(-3) M) in ethylene glycol was significantly higher (ca. 82%) than that of the molar equivalent of NAA content in the product i.e. 0.08 mg in 1×10(-3) M solution. The resulting polymer would be of potential utility as a sustained release plant growth regulator and sensory applications.

  1. Enantioselective Recognition by Chiral Supramolecular Gels.

    PubMed

    Zhang, Li; Jin, Qingxian; Liu, Minghua

    2016-10-06

    Chiral supramolecular gels, in which small organic molecules self-assemble into chiral nanostructures and entangle each other to immobilize solvents through various noncovalent interactions, can work as a matrix for enantioselective recognition on chiral analytes. Through gelation and the formation of well-defined nanostructures, the chiral sense of the component molecules can be accumulated or amplified, and thus, the enantioselective recognition ability can be enhanced. Furthermore, a chiral microenvironment formed in the gel networks could provide additional stereochemical recognition geometry and attribute to efficient recognition. In this focus review, enantioselective recognition on chiral analytes through chiral supramolecular gels, with either amplified signals or the gel-sol phase transition, is discussed. This review is expected to provide useful insights into the design and fabrication of supramolecular gel systems with chiral features and high enantioselectivity.

  2. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    SciTech Connect

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-08-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application.

  3. Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics.

    PubMed

    Geng, Tao; Mathies, Richard A

    2015-01-01

    The presence of PCR inhibitors in forensic and other biological samples reduces the amplification efficiency, sometimes resulting in complete PCR failure. Here we demonstrate a high-performance digital agarose droplet microfluidics technique for single-cell and single-molecule forensic short tandem repeat (STR) typing of samples contaminated with high concentrations of PCR inhibitors. In our multifaceted strategy, the mitigation of inhibitory effects is achieved by the efficient removal of inhibitors from the porous agarose microgel droplets carrying the DNA template through washing and by the significant dilution of targets and remaining inhibitors to the stochastic limit within the ultralow nL volume droplet reactors. Compared to conventional tube-based bulk PCR, our technique shows enhanced (20 ×, 10 ×, and 16 ×) tolerance of urea, tannic acid, and humic acid, respectively, in STR typing of GM09948 human lymphoid cells. STR profiling of single cells is not affected by small soluble molecules like urea and tannic acid because of their effective elimination from the agarose droplets; however, higher molecular weight humic acid still partially inhibits single-cell PCR when the concentration is higher than 200 ng/μL. Nevertheless, the full STR profile of 9948 male genomic DNA contaminated with 500 ng/μL humic acid was generated by pooling and amplifying beads carrying single-molecule 9948 DNA PCR products in a single secondary reaction. This superior performance suggests that our digital agarose droplet microfluidics technology is a promising approach for analyzing low-abundance DNA targets in the presence of inhibitors.

  4. Supplementation of Exogenous Adenosine 5′-Triphosphate Enhances Mechanical Properties of 3D Cell–Agarose Constructs for Cartilage Tissue Engineering

    PubMed Central

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr

    2013-01-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5′-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure–function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct. PMID:23651296

  5. One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

    PubMed

    Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

    2012-08-01

    New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications.

  6. A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate.

    PubMed

    Vashist, Sandeep Kumar; Schneider, E Marion; Luong, John H T

    2015-07-09

    A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL(-1) and 1-243 ng mL(-1), respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.

  7. Xenotransplantation of islets enclosed in agarose microcapsule carrying soluble complement receptor 1.

    PubMed

    Luan, Nguyen Minh; Iwata, Hiroo

    2012-11-01

    Strong immunological reactions remain a major barrier to treating diabetic patients using xenogeneic islets. In a previous study, we developed a method for enclosing islets with agarose microbeads carrying soluble complement receptor 1 (sCR1-Mics), a potent complement inhibitor in both classical and alternative complement activation pathways. This is the follow-up in vivo study to evaluate the protective effect of these sCR1-Mics using a xenotransplantation model (rats to mice). ACI/NSIc rat islets enclosed in sCR1-Mics were transplanted into the intraperitoneal cavity of diabetic C57BL/6 mice without immunosuppression therapy. Transplantation of islets in plain agarose microbeads (Mics) was used as a reference. While islets enclosed in plain Mics were rapidly destroyed (graft survival in recipients of 1000 islets: 11.6±3.8 days), transplantation of islets in sCR1-Mics significantly prolonged graft survival (34.1±3.2 days). Moreover, intraperitoneal glucose tolerance tests revealed that islets in sCR1-Mics normalized blood glucose levels in a similar manner as islets in pancreas of normal mice. In conclusion, sCR1 immobilized onto agarose microbeads exerted some protective effect in xenogeneic islets resulting in prolonged graft survival.

  8. Agarose-assisted micro-contact printing for high-quality biomolecular micro-patterns.

    PubMed

    Jang, Min Jee; Nam, Yoonkey

    2015-05-01

    Micro-contact printing has been developed to print biomolecules, such as cell adhesive molecules, proteins, or DNAs, on a substrate, which can serve as experimental platforms for investigating biological issues and engineering biosensors. Despite the popularity of this method, it has been technically challenging to use a conventional stamp made of a hydrophobic polydimethoxysilane (PDMS) elastomer that often requires surface treatments to facilitate the inking and stamping of biomolecules. In this work, we proposed a new surface modification method for a PDMS stamp using agarose hydrogel and demonstrated the applications to the design of micro-patterned substrates with biomolecules. By using a simple bench-top dip-coating method with a commercial syringe pump to steadily pull out the stamp from boiled agarose solution, we coated an agarose layer on the stamp. It consequentially enhanced the transferability of ink molecules to the target substrate and the uniformity of printed patterns compared to the traditional methods for treating stamp surface such as surfactant coating and temporary oxidation with air plasma. In addition, this microstamping method was also used to produce patterns of proteins with the preservation of bioactivity, which could guide neuronal growth. Thus, we demonstrated the applicability to the interface designs of biochips and biosensors.

  9. Homogeneous tosylation of agarose as an approach toward novel functional polysaccharide materials.

    PubMed

    Gericke, Martin; Heinze, Thomas

    2015-01-01

    The homogeneous tosylation of agarose was studied with respect to the effects of reaction parameters, such as reaction medium, time, and molar ratio, on the reaction course, the degree of substitution (DS) with tosyl/chloro deoxy groups, and the molecular structure. Tosyl agaroses (TOSA) with DS tosyl ≤ 1 .81 could be obtained in completely homogeneous reactions by using N,N-dimethylacetamide (DMA)/LiCl or 1,3-dimethyl-2-imidazolidinone (DMI) as solvents. The products were characterized by FT-IR and NMR spectroscopy and it was demonstrated that two types of substitution pattern can be achieved: (i) non-preferential substitution at position 6 of the 1 → 3-linked β-d-galactose unit (G-6) and position 2 of the 1 → 4-linked 3,6-anyhdro-α-L-galactose unit (LA-2) and (ii) regioselective tosylation at G-6, depending on whether the reaction is performed with or without LiCl. Finally, the nucleophilic displacement reaction of TOSA was studied using azide and ethylenediamine as representative nucleophiles. Novel deoxy-agarose derivatives were obtained that showed an interesting solubility behavior and will be used for creating functional polysaccharide materials.

  10. Chondroitin sulfate-derivatized agarose beads: a new system for studying cation binding to glycosaminoglycans

    SciTech Connect

    Hunter, G.K.

    1987-09-01

    Chondroitin sulfate (CS) has been covalently attached to aminoethyl-agarose beads in a carbodiimide-catalyzed reaction. In this process, an amide bond is formed between carboxylate groups on the glycosaminoglycan (GAG) and the primary amine groups of the beads. Under optimal conditions, up to 160 micrograms of CS is attached per milligram of beads. CS-agarose beads have been used to study Ca binding to GAGs. The beads are mixed with a solution containing CaCl/sub 2/ and /sup 45/Ca and allowed to sediment under unit gravity. An aliquot of supernatant is then removed and /sup 45/Ca activity is determined to quantitate remaining (free) Ca. Using this system, it was shown that CS binds approximately 0.7 Ca/disaccharide unit at saturation. Under the conditions used, the apparent association constant (KA) is approximately 14 mM. In principle, this derivatization protocol may be used to attach any proteoglycan or GAG (except keratan sulfate) to an insoluble support. CS-agarose beads provide a rapid, simple, and relatively artifact-free system for studying cation-GAG interactions.

  11. Aqueous phase catalytic conversion of agarose to 5-hydroxymethylfurfural by metal chlorides

    SciTech Connect

    Yan, Lishi; Laskar, Dhrubojyoti D.; Lee, Suh-Jane; Yang, Bin

    2013-12-14

    Abstract: 5-HMF is a key intermediate for producing chemicals and fuels that can substitute for today’s petroleum-derived feedstocks. A series of metal chlorides, including NaCl, CaCl2, MgCl2, ZnCl2, CuCl2, FeCl3, and CrCl3, were comparatively investigated to catalyze agarose degradation for production of 5-HMF at temperature 180 oC, 200 oC, and 220 oC for 30 min, with catalyst concentration of 0.5% (w/w), 1% (w/w) and 5% (w/w), and substrate concentration of 2% (w/w). Our results revealed that alkali metal chlorides and alkali earth metal chlorides such as NaCl, CaCl2 and MgCl2 gave better 5-HMF yield compared with transition metal chlorides including ZnCl2, CrCl3, CuCl2 and FeCl3. 1% (w/w) MgCl2 was the more favorable catalyst for 5-HMF production from agarose, and resulted in 40.7% 5-HMF yield but no levulinic acid or lactic acid at 200 oC, 35 min. The reaction pathways of agarose degradation catalyzed by MgCl2 were also discussed.

  12. Diffusion of polyelectrolytes in polyelectrolyte gels

    NASA Astrophysics Data System (ADS)

    Rahalkar, Anand; Muthukumar, Murugappan

    2015-03-01

    Using dynamic light scattering, we have investigated the diffusion coefficient of sodium poly(styrene sulfonate) in a matrix of poly(acrylamide-co-acrylate) gels. The diffusion coefficient of the probe polyelectrolyte exhibits a crossover behavior from a particle-diffusion to entropic-barrier dominated diffusion, as the molecular weight is increased. The effect of electrostatics, by varying the charge density of the matrix, on probe diffusion constant will be presented.

  13. Sequence Dependent Electrophoretic Separations of DNA in Pluronic F127 Gels

    NASA Astrophysics Data System (ADS)

    You, Seungyong; van Winkle, David H.

    2010-03-01

    Two-dimensional (2-D) electrophoresis has successfully been used to visualize the separation of DNA fragments of the same length. We electrophorese a double-stranded DNA ladder in an Agarose gel for the first dimension and in gels of Pluronic F127 for the second dimension at room temperature. The 1000 bp band that travels together as a single band in an Agarose gel is split into two bands in Pluronic gels. The slower band follows the exponential decay trend that the other ladder constituents do. After sequencing the DNA fragments, the faster band has an apparently random sequence, while the slower band and the others have two A-tracts in each 250 bp segment. The A-tracts consist of a series of at least five adenine bases pairing with thymine bases. This result leads to the conclusion that the migration of the DNA molecules bent with A-tracts is more retarded in Pluronic gels than the wild-type of DNA molecules.

  14. Matrilysin (MMP-7) is a major matrix metalloproteinase upregulated in biliary atresia-associated liver fibrosis.

    PubMed

    Huang, Chao-Cheng; Chuang, Jiin-Haur; Chou, Ming-Huei; Wu, Chia-Lin; Chen, Ching-Mei; Wang, Chih-Chi; Chen, Yaw-Sen; Chen, Chao-Long; Tai, Ming-Hong

    2005-07-01

    Matrix metalloproteinases (MMPs) are the proteases responsible for tissue remodeling during liver fibrosis caused by various disorders including biliary atresia. However, information regarding the relative contribution of these proteases to liver fibrosis is still limited. We studied matrix metalloproteinase-2 (MMP-2), -7, -9 and -13 mRNA expressions in the liver tissue of early-stage biliary atresia at the time of Kasai's procedure, late-stage biliary atresia at the time of liver transplantation with advanced fibrosis and nondiseased control without liver fibrosis. The results of real-time quantitative reverse transcriptase-PCR analysis revealed that only MMP-2 and -7 expressions were significantly different between groups. MMP-2 was significantly higher in Liver Transplantation group than both in Control (P=0.010) and in Kasai's Procedure (P=0.001) groups, whereas the difference of MMP-2 expression between Control and Kasai's Procedure was not significant. However, the relative expression level of MMP-7 was sequentially elevated when comparing Control, Kasai's Procedure and Liver Transplantation groups, and there was significant (P=0.019) difference when comparing Control and Liver Transplantation groups. Moreover, the fold difference in MMP-7 mRNA was much higher than that in MMP-2 mRNA between groups. The expressions of MMP-7 were further confirmed by agarose gel electrophoresis and Western blotting. Immunohistochemical analysis revealed a significant positive correlation of the scores of MMP-7 immunostaining with the stages of liver fibrosis. In situ hybridization demonstrated that the bile ductular epithelial cells, Kupffer cells and hepatocytes were the major producers of matrix metalloproteinase-7 in the liver. Our results imply that MMP-7 is a major MMP associated with the tissue remodeling during the progression of liver fibrosis in biliary atresia.

  15. Nonlinear elasticity of alginate gels

    NASA Astrophysics Data System (ADS)

    Hashemnejad, Seyed Meysam; Kundu, Santanu

    Alginate is a naturally occurring anionic polysaccharide extracted from brown algae. Because of biocompatibility, low toxicity, and simple gelation process, alginate gels are used in biomedical and food applications. Here, we report the rheological behavior of ionically crosslinked alginate gels, which are obtained by in situ gelation of alginates with calcium salts, in between two parallel plates of a rheometer. Strain stiffening behavior was captured using large amplitude oscillatory shear (LAOS) experiments. In addition, negative normal stress was observed for these gels, which has not been reported earlier for any polysaccharide networks. The magnitude of negative normal stress increases with applied strain and can exceed that of the shear stress at large strain. Rheological results fitted with a constitutive model that considers both stretching and bending of chains indicate that nonlinearity is likely related to the stretching of the chains between the crosslink junctions. The results provide an improved understanding of the deformation mechanism of ionically crosslinked alginate gel and the results will be important in developing synthetic extracellular matrix (ECM) from these materials.

  16. Barriers against DNA-loop formation in a porous matrix

    NASA Astrophysics Data System (ADS)

    Åkerman, Björn

    1996-12-01

    The resistance towards DNA bending imposed by a porous matrix has been investigated by studying the rate of helix-loop penetration through agarose gel pores as driven by electric fields between 1.1 and 6.7 V/cm. YOYO-stained DNA molecules (680 kilo-base-pairs) were prepared in a well-defined globally oriented state by an electrophoretic procedure (YOYO denotes dimer of oxazole-yellow). Loop initiation by a field perpendicular to the global orientation was detected by linear dichroism (LD) spectroscopy in terms of an initial net helix orientation perpendicular to the applied field direction, reflecting the stretching of the chain between the loopholes by the initial growth of the comparatively weakly oriented loop heads. The rate of loop nucleation exhibits a strong field dependence in agreement with a model based on the entropy cost of loop formation. The effect of increasing the average pore radius from 0.7 to 3 P, where P is the persistence length of DNA (500 Å), is significantly weaker than predicted from the model, however. After initially being perpendicular, the net helix orientation is eventually along the field direction, and during this phase the LD exhibits several oscillations before reaching a steady state. By comparison with fluorescence microscopy observations on individual molecules under identical conditions the LD oscillations are identified in terms of loop growth and competition. The spectroscopically measured average rates of these later loop processes exhibit considerably weaker field dependence than loop nucleation, and with power-law dependencies (E1.2-2) in agreement with the DNA coils being stretched by electrophoretic transport of the polymer ends.

  17. Multi-walled carbon nanotube-impregnated agarose film microextraction of polycyclic aromatic hydrocarbons in green tea beverage.

    PubMed

    Loh, Saw Hong; Sanagi, Mohd Marsin; Wan Ibrahim, Wan Aini; Hasan, Mohamed Noor

    2013-03-15

    A new microextraction procedure termed multi-walled carbon nanotube-impregnated agarose film microextraction (MWCNT-AFME) has been developed. The method utilized multi-walled carbon nanotubes (MWCNTs) immobilized in agarose film to serve as adsorbent in solid phase microextraction (SPME). The film was prepared by mixing the MWCNTs in agarose solution and drying the mixture in oven. Extraction of selected polycyclic aromatic hydrocarbons was performed by inserting a needle through circular MWCNT-impregnated agarose films (5 mm diameter) and the assembly was dipped into an agitated sample solution prior to micro high performance liquid chromatography-ultraviolet analysis. Back extraction was then performed using ultrasonication of the films in 100 μL of solvent. The film was discarded after single use, thus avoiding any analyte carry-over effect. Due to the mesoporous nature of the agarose film, the MWCNTs were immobilized easily within the film and thus allowing for close contact between adsorbent and analytes. Under the optimized extraction conditions, the technique achieved trace LODs in the range of 0.1 to 50 ng L(-1) for the targeted analytes, namely fluoranthene, phenanthrene and benzo[a]pyrene. The method was successfully applied to the analysis of spiked green tea beverage samples with good relative recoveries in the range of 91.1 to 107.2%. The results supported the feasibility of agarose to serve as adsorbent holder in SPME which then minimizes the consumption of chemicals and disposal cost of organic wastes.

  18. Fundamentals of gel dosimeters

    NASA Astrophysics Data System (ADS)

    McAuley, K. B.; Nasr, A. T.

    2013-06-01

    Fundamental chemical and physical phenomena that occur in Fricke gel dosimeters, polymer gel dosimeters, micelle gel dosimeters and genipin gel dosimeters are discussed. Fricke gel dosimeters are effective even though their radiation sensitivity depends on oxygen concentration. Oxygen contamination can cause severe problems in polymer gel dosimeters, even when THPC is used. Oxygen leakage must be prevented between manufacturing and irradiation of polymer gels, and internal calibration methods should be used so that contamination problems can be detected. Micelle gel dosimeters are promising due to their favourable diffusion properties. The introduction of micelles to gel dosimetry may open up new areas of dosimetry research wherein a range of water-insoluble radiochromic materials can be explored as reporter molecules.

  19. Microfluidic dielectrophoretic sorter using gel vertical electrodes

    PubMed Central

    Luo, Jason; Nelson, Edward L.; Li, G. P.; Bachman, Mark

    2014-01-01

    We report the development and results of a two-step method for sorting cells and small particles in a microfluidic device. This approach uses a single microfluidic channel that has (1) a microfabricated sieve which efficiently focuses particles into a thin stream, followed by (2) a dielectrophoresis (DEP) section consisting of electrodes along the channel walls for efficient continuous sorting based on dielectric properties of the particles. For our demonstration, the device was constructed of polydimethylsiloxane, bonded to a glass surface, and conductive agarose gel electrodes. Gold traces were used to make electrical connections to the conductive gel. The device had several novel features that aided performance of the sorting. These included a sieving structure that performed continuous displacement of particles into a single stream within the microfluidic channel (improving the performance of downstream DEP, and avoiding the need for additional focusing flow inlets), and DEP electrodes that were the full height of the microfluidic walls (“vertical electrodes”), allowing for improved formation and control of electric field gradients in the microfluidic device. The device was used to sort polymer particles and HeLa cells, demonstrating that this unique combination provides improved capability for continuous DEP sorting of particles in a microfluidic device. PMID:24926390

  20. Nanocrystal/sol-gel nanocomposites

    DOEpatents

    Klimov, Victor L.; Petruska, Melissa A.

    2010-05-25

    The present invention is directed to a process for preparing a solid composite having colloidal nanocrystals dispersed within a sol-gel matrix, the process including admixing colloidal nanocrystals with an amphiphilic polymer including hydrophilic groups selected from the group consisting of --COOH, --OH, --SO.sub.3H, --NH.sub.2, and --PO.sub.3H.sub.2 within a solvent to form an alcohol-soluble colloidal nanocrystal-polymer complex, admixing the alcohol-soluble colloidal nanocrystal-polymer complex and a sol-gel precursor material, and, forming the solid composite from the admixture. The present invention is also directed to the resultant solid composites and to the alcohol-soluble colloidal nanocrystal-polymer complexes.

  1. Preparation of highly luminescent hybrid gel incorporating NAC-capped CdTe quantum dots through sol-gel processing

    NASA Astrophysics Data System (ADS)

    Bu, Hang-Beom; Watanabe, Taichi; Hizume, Masayuki; Takagi, Tomomi; Sobue, Susumu; Kawai, Shoichi; Okuno, Eiichi; Kim, DaeGwi

    2015-03-01

    Highly photoluminescent gel was prepared by embedding water soluble quantum dots (QDs) in an inorganic-organic hybrid gel matrix using a conventional sol-gel process. Aminopropyltrimethoxysilane and citric acid (CA) were found to be the best combination for the gel preparation. 13C-NMR and FT-IR studies indicated hydrogen bond formation between the amine group of APS and the carboxyl group of CA. IR-light radiation curing was comparable to thermal curing and reduced the gelation time to a considerable extent (71 %). The resulting composite formed a hybrid gel phosphor with excellent transparency by embedding CdTe QDs into the matrix and emitted light of various colors with high photoluminescence efficiency (40 %). The gel phosphor retained the PL properties after storage in air for one year. In addition, the strength of the hybrid phosphor was demonstrated by a coin-flipping test.

  2. Gel mesh as "brake" to slow down DNA translocation through solid-state nanopores.

    PubMed

    Tang, Zhipeng; Liang, Zexi; Lu, Bo; Li, Ji; Hu, Rui; Zhao, Qing; Yu, Dapeng

    2015-08-21

    Agarose gel is introduced onto the cis side of silicon nitride nanopores by a simple and low-cost method to slow down the speed of DNA translocation. DNA translocation speed is slowed by roughly an order of magnitude without losing signal to noise ratio for different DNA lengths and applied voltages in gel-meshed nanopores. The existence of the gel moves the center-of-mass position of the DNA conformation further from the nanopore center, contributing to the observed slowing of translocation speed. A reduced velocity fluctuation is also noted, which is beneficial for further applications of gel-meshed nanopores. The reptation model is considered in simulation and agrees well with the experimental results.

  3. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  4. Slow Release Of Reagent Chemicals From Gel Matrices

    NASA Technical Reports Server (NTRS)

    Debnam, William J.; Barber, Patrick G.; Coleman, James

    1988-01-01

    Procedure developed for slow release of reagent chemicals into solutions. Simple and inexpensive and not subject to failure of equipment. Use of toothpaste-type tube or pump dispenser conceivably provides more controlled technique for storage and dispensation of gel matrix. Possible uses include controlled, slow release of reagents in chemical reactions, crystal growth, space-flight experiments, and preformed gel medications from packets.

  5. Formation of Anisotropic Block Copolymer Gels

    NASA Astrophysics Data System (ADS)

    Liaw, Chya Yan; Shull, Kenneth; Henderson, Kevin; Joester, Derk

    2011-03-01

    Anisotropic, fibrillar gels are important in a variety of processes. Biomineralization is one example, where the mineralization process often occurs within a matrix of collagen or chitin fibers that trap the mineral precursors and direct the mineralization process. We wish to replicate this type of behavior within block copolymer gels. Particularly, we are interested in employing gels composed of cylindrical micelles, which are anisotropic and closely mimic biological fibers. Micelle geometry is controlled in our system by manipulating the ratio of molecular weights of the two blocks and by controlling the detailed thermal processing history of the copolymer solutions. Small-Angle X-ray Scattering and Dynamic Light Scattering are used to determine the temperature dependence of the gel formation process. Initial experiments are based on a thermally-reversible alcohol-soluble system, that can be subsequently converted to a water soluble system by hydrolysis of a poly(t-butyl methacrylate) block to a poly (methacrylic acid) block. MRSEC.

  6. A least-squares error minimization approach in the determination of ferric ion diffusion coefficient of Fricke-infused dosimeter gels

    SciTech Connect

    Tseng, Y.J.; Huang, S.-C.; Chu, W.C.

    2005-04-01

    A least-squares error minimization approach was adopted to assess ferric ion diffusion coefficient of Fricke-agarose gels. Ferric ion diffusion process was modeled as a Gaussian-shaped degradation kernel operating on an initial concentration distribution. Diffusion coefficient was iteratively determined by minimizing the error function defined as the difference between the theoretically calculated and the experimentally measured dose distributions. A rapid MR image-based differential gel dosimetry technique that time resolves the evolution of the ferric ion diffusion process minimizes smearing of the dose distribution. Our results showed that for a Fricke-agarose gel contained 1 mM ammonium ferrous sulfate, 1% agarose, 1 mM sodium chloride, and 50 mM sulfuric acid, its ferric ion diffusion coefficient is (1.59{+-}0.28)x10{sup -2} cm{sup 2} h{sup -1} at room temperature. This value falls within the 1.00-2.00x10{sup -2} cm{sup 2} h{sup -1} range previously reported under varying gelling ingredients and concentrations. This method allows a quick, nondestructive evaluation of the ferric ion diffusion coefficient that can be used in conjunction with the in situ gel dosimetry experiment to provide a practical diffusion characterization of the dosimeter gel.

  7. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

    PubMed

    Cuttitta, Christina M; Ericson, Daniel L; Scalia, Alexander; Roessler, Christian G; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M; Sweet, Robert M; Soares, Alexei S

    2015-01-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  8. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    SciTech Connect

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  9. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    DOE PAGES

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; ...

    2014-06-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s-1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from themore » inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.« less

  10. Rheological characterization of human fibrin and fibrin-agarose oral mucosa substitutes generated by tissue engineering.

    PubMed

    Rodríguez, I A; López-López, M T; Oliveira, A C X; Sánchez-Quevedo, M C; Campos, A; Alaminos, M; Durán, J D G

    2012-08-01

    In regenerative medicine, the generation of biocompatible substitutes of tissues by in vitro tissue engineering must fulfil certain requirements. In the case of human oral mucosa, the rheological properties of tissues deserve special attention because of their influence in the acoustics and biomechanics of voice production. This work is devoted to the rheological characterization of substitutes of the connective tissue of the human oral mucosa. Two substitutes, composed of fibrin and fibrin-agarose, were prepared in cell culture for periods in the range 1-21 days. The time evolution of the rheological properties of both substitutes was studied by two different experimental procedures: steady-state and oscillatory measurements. The former allows the plastic behaviour of the substitutes to be characterized by estimating their yield stress; the latter is employed to quantify their viscoelastic responses by obtaining the elastic (G') and viscous (G'') moduli. The results demonstrate that both substitutes are characterized by a predominant elastic response, in which G' (order 100 Pa) is roughly one order of magnitude larger than G'' (order 10 Pa). But the most relevant insight is the stability, throughout the 21 days of culture time, of the rheological quantities in the case of fibrin-agarose, whereas the fibrin substitute shows a significant hardening. This result provides evidence that the addition to fibrin of a small amount of agarose allows the rheological stability of the oral mucosa substitute to be maintained. This feature, together with its viscoelastic similitude with native tissues, makes this biomaterial appropriate for potential use as a scaffold in regenerative therapies of human oral mucosa.

  11. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    SciTech Connect

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2014-06-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s-1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  12. Oriented Attachment of Recombinant Proteins to Agarose-Coated Magnetic Nanoparticles by Means of a β-Trefoil Lectin Domain.

    PubMed

    Acebrón, Iván; Ruiz-Estrada, Amalia G; Luengo, Yurena; Morales, María Del Puerto; Guisán, José Manuel; Mancheño, José Miguel

    2016-11-16

    Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currently represents an active field of research. With this in mind, we have prepared and characterized agarose-coated maghemite nanoparticles to set up a platform for the attachment of recombinant proteins fused to the β-trefoil lectin domain LSL150, a small protein that combines fusion tag properties with agarose-binding capacity. Analysis of the agarose-coated nanoparticles by dynamic light scattering, Fourier transform infrared spectroscopy, and thermogravimetric studies shows that decoupling particle formation from agarose coating provides better results in terms of coating efficiency and particle size distribution. LSL150 interacts with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes, which in turn can be dissociated ad hoc with the competing sugar lactose. Characterization of the complexes formed with the fusion proteins LSL-EGFP (LSL-tagged enhanced green fluorescent protein from Aquorea victoria) and LSL-BTL2 (LSL-tagged lipase from Geobacillus thermocatenolatus) provided evidence supporting a topologically oriented binding of these molecules to the interface of the agarose-coated nanoparticles. This is consistent with the marked polarity of the β-trefoil structure where the sugar-binding sites and the N- and C-terminus ends are at opposed sides. In summary, LSL150 displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles.

  13. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    PubMed Central

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  14. Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.

    PubMed

    Mohannath, Gireesha; Pikaard, Craig S

    2016-01-01

    Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb-9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

  15. Isolation of hen egg white lysozyme, ovotransferrin and ovalbumin, using a quaternary ammonium bound to a highly crosslinked agarose matrix.

    PubMed

    Vachier, M C; Piot, M; Awadé, A C

    1995-02-03

    A single-step anion-exchange chromatographic separation of egg white proteins was carried out using a Q Sepharose Fast Flow column. The separation resulted in the isolation of two lysozyme peaks with purities of ca. 99 and 88%, one peak of ovotransferrin purified to ca. 75% and two ovalbumin peaks with purities of ca. 54 and 98%. Recoveries were estimated to be ca. 60, 100 and 83% for lysozyme, ovotransferrin and ovalbumin, respectively. The amino acid compositions of all collected peaks have also been determined. This confirmed the identity of some of the proteins contained in these peaks.

  16. Surfactant free fractions of metallic and semiconducting single-walled carbon nanotubes via optimised gel chromatography

    SciTech Connect

    Lukaszczuk, Pawel; Ruemmeli, Mark H.; Knupfer, Martin; Kalenczuk, Ryszard J.; Borowiak-Palen, Ewa

    2012-03-15

    Highlights: Black-Right-Pointing-Pointer The application of gel permeation chromatography technique in a field of SWCNT separation. Black-Right-Pointing-Pointer Non-commercial agarose gel used as a column filling. Black-Right-Pointing-Pointer Purification route is presented, quality and quantity estimation is shown. Black-Right-Pointing-Pointer Process is ready for high-scale separation of SWCNTs. -- Abstract: We report the procedure of sorting/purification of carbon nanotubes by electronic type using chromatographic column with sodium dodecylsulfate (SDS) and sodium deoxycholate (DOC) solutions as the eluents. The non-commercial agarose gel in different concentrations has been tested in the process. It was found that in optimal gel concentration the fractionation resulted in {approx}96.2% yield of semiconducting species. Importantly, to get surfactant-free fractions the post-separation purification procedure has been carried out. The UV-vis-NIR and Raman spectroscopy have been utilised for the samples analysis. High resolution transmission microscopy and thermogravimetric analysis allowed to study the sample morphology and purity, respectively.

  17. Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus.

    PubMed

    Smejkal, Gary B; Shainoff, John R; Kottke-Marchant, Kandice M

    2003-02-01

    Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.5 degrees C. At 10 degrees C operative temperature, NRAGE at 1.5% agarose concentration was completed in 20 min at 250 V. Electrophoresis could be performed in only 10 min at 500 V, but at such high voltages, localized temperature fluctuations as much as 6 degrees C resulted in perturbation of banding patterns in those vicinities. In the optimized method, both high molecular weight multimers and proteolytic fragments of vWF were separable suggesting clinical applicability of this system for the diagnosis of von Willebrand Disease and thrombotic thrombocytopenic purpura.

  18. Effect of the hydration on the biomechanical properties in a fibrin-agarose tissue-like model.

    PubMed

    Scionti, Giuseppe; Moral, Monica; Toledano, Manuel; Osorio, Raquel; Durán, Juan D G; Alaminos, Miguel; Campos, Antonio; López-López, Modesto T

    2014-08-01

    The effect of hydration on the biomechanical properties of fibrin and fibrin-agarose (FA) tissue-like hydrogels is reported. Native hydrogels with approximately 99.5% of water content and hydrogels with water content reduced until 90% and 80% by means of plastic compression (nanostructuration) were generated. The biomechanical properties of the hydrogels were investigated by tensile, compressive, and shear tests. Experimental results indicate that nanostructuration enhances the biomechanical properties of the hydrogels. This improvement is due to the partial draining of the water that fills the porous network of fibers that the plastic compression generates, which produces a denser material, as confirmed by scanning electron microscopy. Results also indicate that the characteristic compressive and shear parameters increase with agarose concentration, very likely due to the high water holding capacity of agarose, which reduces the compressibility and gives consistency to the hydrogels. However, results of tensile tests indicate a weakening of the hydrogels as agarose concentration increases, which evidences the anisotropic nature of these biomaterials. Interestingly, we found that by adjusting the water and agarose contents it is possible to tune the biomechanical properties of FA hydrogels for a broad range, within which the properties of many native tissues fall.

  19. Silica scintillating materials prepared by sol-gel methods

    SciTech Connect

    Werst, D.W.; Sauer, M.C. Jr.; Cromack, K.R.; Lin, Y.; Tartakovsky, E.A.; Trifunac, A.D.

    1993-12-31

    Silica was investigated as a rad-hard alternative to organic polymer hosts for organic scintillators. Silica sol-gels were prepared by hydrolysis of tetramethoxysilane in alcohol solutions. organic dyes were incorporated into the gels by dissolving in methanol at the sol stage of gel formation. The silica sol-gel matrix is very rad-hard. The radiation stability of silica scintillators prepared by this method is dye-limited. Transient radioluminescence was measured following excitation with 30 ps pulses of 20 MeV electrons.

  20. Molecular transport in collagenous tissues measured by gel electrophoresis.

    PubMed

    Hunckler, Michael D; Tilley, Jennifer M R; Roeder, Ryan K

    2015-11-26

    Molecular transport in tissues is important for drug delivery, nutrient supply, waste removal, cell signaling, and detecting tissue degeneration. Therefore, the objective of this study was to investigate gel electrophoresis as a simple method to measure molecular transport in collagenous tissues. The electrophoretic mobility of charged molecules in tissue samples was measured from relative differences in the velocity of a cationic dye passing through an agarose gel in the absence and presence of a tissue section embedded within the gel. Differences in electrophoretic mobility were measured for the transport of a molecule through different tissues and tissue anisotropy, or the transport of different sized molecules through the same tissue. Tissue samples included tendon and fibrocartilage from the proximal (tensile) and distal (compressive) regions of the bovine flexor tendon, respectively, and bovine articular cartilage. The measured electrophoretic mobility was greatest in the compressive region of the tendon (fibrocartilage), followed by the tensile region of tendon, and lowest in articular cartilage, reflecting differences in the composition and organization of the tissues. The anisotropy of tendon was measured by greater electrophoretic mobility parallel compared with perpendicular to the predominate collagen fiber orientation. Electrophoretic mobility also decreased with increased molecular size, as expected. Therefore, the results of this study suggest that gel electrophoresis may be a useful method to measure differences in molecular transport within various tissues, including the effects of tissue type, tissue anisotropy, and molecular size.

  1. Use of leucocyte migration under agarose to study spontaneous and directed locomotion of leucocytes.

    PubMed Central

    Repo, H; Kostiala, A A; Kosunen, T U

    1978-01-01

    Three different cell attractants, together with the parallel use of the leucocyte migration agarose test (LMAT) and the leading front modification (LFM) of the Boyden chamber technique, were employed in studying whether the maximal migration of normal human polymorphonuclear leucocytes (PMNs) is higher toward an attractant (chemotaxis) than in the same attractant incorporated in the culture media (chemokinesis). Using LMAT, the maximal migration distance toward zymosan activated serum (ZAS) was found to be significantly longer than that under agarose mixed with ZAS, thus indicating a chemotactic effect exerted by ZAS. When bacterial culture filtrate (BCF) and casein were used as attractants, the corresponding difference was not significant, implying that the stimulatory effect of these substances on cell migration could be explained by increased random locomotion (chemokinesis) alone. In LFM, the migration rate was significantly higher along a casein gradient than without a gradient. Using ZAS, however, only chemokinesis could be demonstrated. BCF was found to attract PMNs into membrane filters only in the presence of human serum albumin. These observations give credence to the view that both LMAT and LFM are applicable to the in vitro assessment of chemotaxis and chemokinesis but the attractant of choice for this is different in each of the two methods. Images Figure 1 PMID:359465

  2. Cell fractionation with affinity ligands conjugated to agarose-polyacrolein microsphere beads.

    PubMed

    Margel, S; Ofarim, M; Eshhar, Z

    1983-07-01

    A new effective insoluble support useful for cell fractionation based on agarose-polyacrolein microsphere beads (APAMB) of diameters 150--250 micrometers has been developed. The synthesized polyacrolein (PA) microspheres, of average diameter 0.2 micrometer, are provided with reactive aldehyde groups through which various ligands containing primary amino groups are bound covalently in a single step at physiological pH. Antibodies coupled to the microspheres are very effective for labelling of cell surface receptors on human red blood cells and mouse lymphoid cells. APAMB were obtained by encapsulating the PA microspheres with agarose. Antibodies and lectins bound to the APAMB serve to construct affinity columns for the separation of red blood cells and murine lymphocyte subpopulations. Anti-human red blood cell antibodies coupled to anti-immunoglobulin APAMB are effective in separating human from turkey red blood cells, whereas either anti-Thy 1.2 anti-immunoglobulin antibodies or soybean agglutinin coupled to APAMB have proved useful for the separation of T and B cells from heterogeneous population of spleen cells. The separation procedure is simple, rapid and effective. The viability of the fractionated cells is unaffected by the procedure and the recovery of the cells is high: between 80% and 100%.

  3. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

  4. A Novel Agarolytic β-Galactosidase Acts on Agarooligosaccharides for Complete Hydrolysis of Agarose into Monomers

    PubMed Central

    Lee, Chan Hyoung; Kim, Hee Taek; Yun, Eun Ju; Lee, Ah Reum; Kim, Sa Rang; Kim, Jae-Han; Choi, In-Geol

    2014-01-01

    Marine red macroalgae have emerged to be renewable biomass for the production of chemicals and biofuels, because carbohydrates that form the major component of red macroalgae can be hydrolyzed into fermentable sugars. The main carbohydrate in red algae is agarose, and it is composed of d-galactose and 3,6-anhydro-l-galactose (AHG), which are alternately bonded by β1-4 and α1-3 linkages. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium (Vibrio sp. strain EJY3); the enzyme is annotated as Vibrio sp. EJY3 agarolytic β-galactosidase (VejABG). Unlike the lacZ-encoded β-galactosidase from Escherichia coli, VejABG does not hydrolyze common substrates like lactose and can act only on the galactose moiety at the nonreducing end of AOS. The optimum pH and temperature of VejABG on an agarotriose substrate were 7 and 35°C, respectively. Its catalytic efficiency with agarotriose was also similar to that with agaropentaose or agaroheptaose. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of this novel β-galactosidase offers an enormous advantage in the saccharification of agarose or agar in red macroalgae for its use as a biomass feedstock for fermentable sugar production. PMID:25038102

  5. Simulated moving bed separation of agarose-hydrolyzate components for biofuel production from marine biomass.

    PubMed

    Kim, Pung-Ho; Nam, Hee-Geun; Park, Chanhun; Wang, Nien-Hwa Linda; Chang, Yong Keun; Mun, Sungyong

    2015-08-07

    The economically-efficient separation of galactose, levulinic acid (LA), and 5-hydroxymethylfurfural (5-HMF) in acid hydrolyzate of agarose has been a key issue in the area of biofuel production from marine biomass. To address this issue, an optimal simulated moving bed (SMB) process for continuous separation of the three agarose-hydrolyzate components with high purities, high yields, and high throughput was developed in this study. As a first step for this task, the adsorption isotherm and mass-transfer parameters of each component on the qualified adsorbent were determined through a series of multiple frontal experiments. The determined parameters were then used in optimizing the SMB process for the considered separation. Finally, the optimized SMB process was tested experimentally using a self-assembled SMB unit with four zones. The SMB experimental results and the relevant computer simulations verified that the developed process in this study was quite successful in the economically-efficient separation of galactose, LA, and 5-HMF in a continuous mode with high purities and high yields. It is thus expected that the developed SMB process in this study will be able to serve as one of the trustworthy ways of improving the economic feasibility of biofuel production from marine biomass.

  6. Location of Biomarkers and Reagents within Agarose Beads of a Programmable Bio-nano-chip

    PubMed Central

    Jokerst, Jesse V.; Chou, Jie; Camp, James P.; Wong, Jorge; Lennart, Alexis; Pollard, Amanda A.; Floriano, Pierre N.; Christodoulides, Nicolaos; Simmons, Glennon W.; Zhou, Yanjie; Ali, Mehnaaz F.

    2012-01-01

    The slow development of cost-effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable bio-nano-chip (PBNC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PBNC system employs ≈300 μm-diameter bead sensors composed of agarose “nanonets” that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein-capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead-bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered. PMID:21290601

  7. Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds.

    PubMed

    Puhakka, P H; Ylärinne, J H; Lammi, M J; Saarakkala, S; Tiitu, V; Kröger, H; Virén, T; Jurvelin, J S; Töyräs, J

    2014-11-07

    Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT.

  8. Covalent attachment of lipases on glyoxyl-agarose beads: application in fruit flavor and biodiesel synthesis.

    PubMed

    Mendes, Adriano A; de Castro, Heizir F; Giordano, Raquel L C

    2014-09-01

    The aim of this work was to prepare biocatalysts to catalyze the synthesis of butyl butyrate by esterification reaction, and the synthesis of biodiesel by transesterification of palm and babassu oils with ethanol. Lipase preparations Lipolase® (TLL1) and Lipex® 100 L (TLL2) from Thermomyces lanuginosus and Lipase AK from Pseudomonas fluorescens (PFL) were immobilized on glyoxyl-agarose beads prepared by activation with glycidol (Gly) and epichlorohydrin (Epi). The influence of immobilization time, lipase source and activating agents on the catalytic activity of the biocatalysts were evaluated in both aqueous and organic media. TLL1 immobilized on glyoxyl-agarose by 24 h of incubation resulted biocatalysts with high hydrolytic activity (varying from 1347.3 to 1470.0 IU/g of support) and thermal-stability, around 300-fold more stable than crude TLL1 extract. The maximum load of immobilized TLL1 was around 20 mg of protein/g of support. The biocatalyst prepared exhibited high activity and operational stability on the butyl butyrate synthesis by esterification after five successive cycles of 24 h each (conversion around 85-90%). Immobilized TLL1 and PFL were active in the synthesis of biodiesel by transesterification reaction. Maximum transesterification yield (≥98.5% after 48 h of reaction at 45°C) was provided by using palm oil as feedstock.

  9. The Sol-Gel-Xerogel Transition

    DTIC Science & Technology

    1993-11-01

    cases, on the matrix of the gel. They showed that photofading of methylene blue in thin films prepared from methyltriethoxy-silane is faster than In films...thin films doped with zeolite crystals (>1 pim, ZSM-5) benefit from the size exclusion selectivity of the encased zeolites . 2. Control of the surface...exposed to H2S due to the formation of CdS crystals. Zink and 0 Dunn [33] reported that sol-gel glasses doped with iron(III) produce an intense blue

  10. Hybrid sol-gel optical materials

    DOEpatents

    Zeigler, J.M.

    1993-04-20

    Hybrid sol-gel materials comprise silicate sols cross-linked with linear polysilane, polygermane, or poly(silane-germane). The sol-gel materials are useful as optical identifiers in tagging and verification applications and, in a different aspect, as stable, visible light transparent non-linear optical materials. Methyl or phenyl silicones, polyaryl sulfides, polyaryl ethers, and rubbery polysilanes may be used in addition to the linear polysilane. The linear polymers cross-link with the sol to form a matrix having high optical transparency, resistance to thermooxidative aging, adherence to a variety of substrates, brittleness, and a resistance to cracking during thermal cycling.

  11. Hybrid sol-gel optical materials

    DOEpatents

    Zeigler, John M.

    1993-01-01

    Hybrid sol-gel materials comprise silicate sols cross-linked with linear polysilane, polygermane, or poly(silane-germane). The sol-gel materials are useful as optical identifiers in tagging and verification applications and, in a different aspect, as stable, visible light transparent non-linear optical materials. Methyl or phenyl silicones, polyaryl sulfides, polyaryl ethers, and rubbery polysilanes may be used in addition to the linear polysilane. The linear polymers cross-link with the sol to form a matrix having high optical transparency, resistance to thermooxidative aging, adherence to a variety of substrates, brittleness, and a resistance to cracking during thermal cycling.

  12. Hybrid sol-gel optical materials

    DOEpatents

    Zeigler, John M.

    1992-01-01

    Hybrid sol-gel materials comprise silicate sols cross-linked with linear polysilane, polygermane, or poly(silane-germane). The sol-gel materials are useful as optical identifiers in tagging and verification applications and, in a different aspect, as stable, visible light transparent non-linear optical materials. Methyl or phenyl silicones, polyaryl sulfides, polyaryl ethers, and rubbery polysilanes may be used in addition to the linear polysilane. The linear polymers cross-link with the sol to form a matrix having high optical transparency, resistance to thermooxidative aging, adherence to a variety of substrates, brittleness, and a resistance to cracking during thermal cycling.

  13. Functionalizing single crystals: incorporation of nanoparticles inside gel-grown calcite crystals.

    PubMed

    Liu, Yujing; Yuan, Wentao; Shi, Ye; Chen, Xiaoqiang; Wang, Yong; Chen, Hongzheng; Li, Hanying

    2014-04-14

    Synthetic single crystals are usually homogeneous solids. Biogenic single crystals, however, can incorporate biomacromolecules and become inhomogeneous solids so that their properties are also extrinsically regulated by the incorporated materials. The discrepancy between the properties of synthetic and biogenic single crystals leads to the idea to modify the internal structure of synthetic crystals to achieve nonintrinsic properties by incorporation of foreign material. Intrinsically colorless and diamagnetic calcite single crystals are turned into colored and paramagnetic solids, through incorporation of Au and Fe3O4 nanoparticles without significantly disrupting the crystalline lattice of calcite. The crystals incorporate the nanoparticles and gel fibers when grown in agarose gel media containing the nanoparticles, whereas the solution-grown crystals do not. As such, our work extends the long-history gel method for crystallization into a platform to functionalize single-crystalline materials.

  14. Band broadening in gel electrophoresis: scaling laws for the dispersion coefficient measured by FRAP.

    PubMed

    Tinland, B; Pernodet, N; Pluen, A

    1998-10-05

    We determined quantitatively the band broadening effect during gel electrophoresis by measuring the longitudinal dispersion coefficient Dx, with a fluorescence recovery after photobleaching setup, coupled to an electrophoretic cell. We carried out measurements as a function of the electric field, the average pore size, and the molecular length of DNA fragments. Our results are in good agreement with the predictions of the biased reptation model with fluctuations described by T. A. Duke et al. [(1992) Physics Review Letters, vol. 69, pp. 3260-3263]. This agreement is observed on single-stranded DNA [persistence length approximately equal to 4 nm; B. Tinland et al. (1997) Macromolecules, vol. 30, pp. 5763-5765] in polyacrylamide gels and on double-stranded DNA (persistence length approximately equal to 50 nm) in agarose gels, two systems where the ratio between the average pore size and the Kuhn length is larger than 1.

  15. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

    PubMed

    Laskey, R A; Mills, A D

    1975-08-15

    Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

  16. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2003-09-01

    This report describes work performed during the second year of the project, ''Conformance Improvement Using Gels.'' The project has two objectives. The first objective is to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective is to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil. Pore-level images from X-ray computed microtomography were re-examined for Berea sandstone and porous polyethylene. This analysis suggests that oil penetration through gel-filled pores occurs by a gel-dehydration mechanism, rather than a gel-ripping mechanism. This finding helps to explain why aqueous gels can reduce permeability to water more than to oil. We analyzed a Cr(III)-acetate-HPAM gel treatment in a production well in the Arbuckle formation. The availability of accurate pressure data before, during, and after the treatment was critical for the analysis. After the gel treatment, water productivity was fairly constant at about 20% of the pre-treatment value. However, oil productivity was stimulated by a factor of 18 immediately after the treatment. During the six months after the treatment, oil productivity gradually decreased to approach the pre-treatment value. To explain this behavior, we proposed that the fracture area open to oil flow was increased substantially by the gel treatment, followed by a gradual closing of the fractures during subsequent production. For a conventional Cr(III)-acetate-HPAM gel, the delay between gelant preparation and injection into a fracture impacts the placement, leakoff, and permeability reduction behavior. Formulations placed as partially formed gels showed relatively low pressure gradients during placement, and yet substantially reduced the flow capacity of

  17. Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions.

    PubMed

    Azqueta, Amaya; Gutzkow, Kristine B; Brunborg, Gunnar; Collins, Andrew R

    2011-09-18

    The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.

  18. Bioactive Agarose Carbon-Nanotube Composites are Capable of Manipulating Brain–Implant Interface

    PubMed Central

    Lewitus, Dan Y.; Smith, Karen L.; Landers, John; Neimark, Alexander V.; Kohn, Joachim

    2014-01-01

    Composite electrodes made of the polysaccharide agarose and carbon nanotube fibers (A-CNE) have shown potential to be applied as tissue-compatible, micro-electronic devices. In the present work, A-CNEs were functionalized using neuro-relevant proteins (laminin and alpha-melanocyte stimulating hormone) and implanted in brain tissue for 1 week (acute response) and 4 weeks (chronic response). Qualitative and quantitative analysis of neuronal and immunological responses revealed significant changes in immunological response to implanted materials depending on the type of biomolecule used. The potential to manipulate tissue response through the use of an anti-inflammatory protein, alpha-melanocyte stimulating hormone, was shown in the reduction of astroglia presence near the implant site during the glial scar formation. These results suggest that A-CNEs, which are soft, flexible, and easily made bioactive, have the ability to modify brain tissue response through surface modification as a function of the biomolecule used. PMID:25382868

  19. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2004-09-30

    This report describes work performed during the third and final year of the project, ''Conformance Improvement Using Gels.'' Corefloods revealed throughput dependencies of permeability reduction by polymers and gels that were much more prolonged during oil flow than water flow. This behavior was explained using simple mobility ratio arguments. A model was developed that quantitatively fits the results and predicts ''clean up'' times for oil productivity when production wells are returned to service after application of a polymer or gel treatment. X-ray computed microtomography studies of gels in strongly water-wet Berea sandstone and strongly oil-wet porous polyethylene suggested that oil penetration through gel-filled pores occurs by a gel-dehydration mechanism, rather than gel-ripping or gel-displacement mechanisms. In contrast, analysis of data from the University of Kansas suggests that the gel-ripping or displacement mechanisms are more important in more permeable, strongly water-wet sandpacks. These findings help to explain why aqueous gels can reduce permeability to water more than to oil under different conditions. Since cement is the most commonly used material for water shutoff, we considered when gels are preferred over cements. Our analysis and experimental results indicated that cement cannot be expected to completely fill (top to bottom) a vertical fracture of any width, except near the wellbore. For vertical fractures with apertures less than 4 mm, the cement slurry will simply not penetrate very far into the fracture. For vertical fractures with apertures greater than 4 mm, the slurry may penetrate a substantial distance into the bottom part of the fracture. However, except near the wellbore, the upper part of the fracture will remain open due to gravity segregation. We compared various approaches to plugging fractures using gels, including (1) varying polymer content, (2) varying placement (extrusion) rate, (3) using partially formed gels, (4

  20. Comparison of potassium and sodium binding in vivo and in agarose samples using TQTPPI pulse sequence

    NASA Astrophysics Data System (ADS)

    Schepkin, Victor D.; Neubauer, Andreas; Nagel, Armin M.; Budinger, Thomas F.

    2017-04-01

    Potassium and sodium specific binding in vivo were explored at 21.1 T by triple quantum (TQ) magnetic resonance (MR) signals without filtration to achieve high sensitivities and precise quantifications. The pulse sequence used time proportional phase increments (TPPI). During simultaneous phase-time increments, it provided total single quantum (SQ) and TQ MR signals in the second dimension at single and triple quantum frequencies, respectively. The detection of both TQ and SQ signals was performed at identical experimental conditions and the resulting TQ signal equals 60 ± 3% of the SQ signal when all ions experience sufficient time for binding. In a rat head in vivo the TQ percentage relative to SQ for potassium is 41.5 ± 3% and for sodium is 16.1 ± 1%. These percentages were compared to the matching values in an agarose tissue model with MR relaxation times similar to those of mammalian brain tissue. The sodium TQ signal in agarose samples decreased in the presence of potassium, suggesting a competitive binding of potassium relative to sodium ions for the same binding sites. The TQTPPI signals correspond to almost two times more effective binding of potassium than sodium. In vivo, up to ∼69% of total potassium and ∼27% of total sodium can be regarded as bound or experiencing an association time in the range of several milliseconds. Experimental data analyses show that more than half of the in vivo total sodium TQ signal could be from extracellular space, which is an important factor for quantification of intracellular MR signals.

  1. Carbon dots rooted agarose hydrogel hybrid platform for optical detection and separation of heavy metal ions.

    PubMed

    Gogoi, Neelam; Barooah, Mayuri; Majumdar, Gitanjali; Chowdhury, Devasish

    2015-02-11

    A robust solid sensing platform for an on-site operational and accurate detection of heavy metal is still a challenge. We introduce chitosan based carbon dots rooted agarose hydrogel film as a hybrid solid sensing platform for detection of heavy metal ions. The fabrication of the solid sensing platform is centered on simple electrostatic interaction between the NH3+ group present in the carbon dots and the OH- groups present in agarose. Simply on dipping the hydrogel film strip into the heavy metal ion solution, in particular Cr6+, Cu2+, Fe3+, Pb2+, Mn2+, the strip displays a color change, viz., Cr6+→yellow, Cu2+→blue, Fe3+→brown, Pb2+→white, Mn2+→tan brown. The optical detection limit of the respective metal ion is found to be 1 pM for Cr6+, 0.5 μM for Cu2+, and 0.5 nM for Fe3+, Pb2+, and Mn2+ by studying the changes in UV-visible reflectance spectrum of the hydrogel film. Moreover, the hydrogel film finds applicability as an efficient filtration membrane for separation of these quintet heavy metal ions. The strategic fundamental feature of this sensing platform is the successful capability of chitosan to form colored chelates with transition metals. This proficient hybrid hydrogel solid sensing platform is thus the most suitable to employ as an on-site operational, portable, cheap colorimetric-optical detector of heavy metal ion with potential skill in their separation. Details of the possible mechanistic insight into the colorimetric detection and ion separation are also discussed.

  2. Comparative assessment of intrinsic mechanical stimuli on knee cartilage and compressed agarose constructs.

    PubMed

    Completo, A; Bandeiras, C; Fonseca, F

    2017-03-17

    A well-established cue for improving the properties of tissue-engineered cartilage is mechanical stimulation. However, the explicit ranges of mechanical stimuli that correspond to favorable metabolic outcomes are elusive. Usually, these outcomes have only been associated with the applied strain and frequency, an oversimplification that can hide the fundamental relationship between the intrinsic mechanical stimuli and the metabolic outcomes. This highlights two important key issues: the firstly is related to the evaluation of the intrinsic mechanical stimuli of native cartilage; the second, assuming that the intrinsic mechanical stimuli will be important, deals with the ability to replicate them on the tissue-engineered constructs. This study quantifies and compares the volume of cartilage and agarose subjected to a given magnitude range of each intrinsic mechanical stimulus, through a numerical simulation of a patient-specific knee model coupled with experimental data of contact during the stance phase of gait, and agarose constructs under direct-dynamic compression. The results suggest that direct compression loading needs to be parameterized with time-dependence during the initial culture period in order to better reproduce each one of the intrinsic mechanical stimuli developed in the patient-specific cartilage. A loading regime which combines time periods of low compressive strain (5%) and frequency (0.5Hz), in order to approach the maximal principal strain and fluid velocity stimulus of the patient-specific cartilage, with time periods of high compressive strain (20%) and frequency (3Hz), in order to approach the pore pressure values, may be advantageous relatively to a single loading regime throughout the full culture period.

  3. Ultraflexible organic amplifier with biocompatible gel electrodes

    NASA Astrophysics Data System (ADS)

    Sekitani, Tsuyoshi; Yokota, Tomoyuki; Kuribara, Kazunori; Kaltenbrunner, Martin; Fukushima, Takanori; Inoue, Yusuke; Sekino, Masaki; Isoyama, Takashi; Abe, Yusuke; Onodera, Hiroshi; Someya, Takao

    2016-04-01

    In vivo electronic monitoring systems are promising technology to obtain biosignals with high spatiotemporal resolution and sensitivity. Here we demonstrate the fabrication of a biocompatible highly conductive gel composite comprising multi-walled carbon nanotube-dispersed sheet with an aqueous hydrogel. This gel composite exhibits admittance of 100 mS cm-2 and maintains high admittance even in a low-frequency range. On implantation into a living hypodermal tissue for 4 weeks, it showed a small foreign-body reaction compared with widely used metal electrodes. Capitalizing on the multi-functional gel composite, we fabricated an ultrathin and mechanically flexible organic active matrix amplifier on a 1.2-μm-thick polyethylene-naphthalate film to amplify (amplification factor: ~200) weak biosignals. The composite was integrated to the amplifier to realize a direct lead epicardial electrocardiography that is easily spread over an uneven heart tissue.

  4. Ultraflexible organic amplifier with biocompatible gel electrodes.

    PubMed

    Sekitani, Tsuyoshi; Yokota, Tomoyuki; Kuribara, Kazunori; Kaltenbrunner, Martin; Fukushima, Takanori; Inoue, Yusuke; Sekino, Masaki; Isoyama, Takashi; Abe, Yusuke; Onodera, Hiroshi; Someya, Takao

    2016-04-29

    In vivo electronic monitoring systems are promising technology to obtain biosignals with high spatiotemporal resolution and sensitivity. Here we demonstrate the fabrication of a biocompatible highly conductive gel composite comprising multi-walled carbon nanotube-dispersed sheet with an aqueous hydrogel. This gel composite exhibits admittance of 100 mS cm(-2) and maintains high admittance even in a low-frequency range. On implantation into a living hypodermal tissue for 4 weeks, it showed a small foreign-body reaction compared with widely used metal electrodes. Capitalizing on the multi-functional gel composite, we fabricated an ultrathin and mechanically flexible organic active matrix amplifier on a 1.2-μm-thick polyethylene-naphthalate film to amplify (amplification factor: ∼200) weak biosignals. The composite was integrated to the amplifier to realize a direct lead epicardial electrocardiography that is easily spread over an uneven heart tissue.

  5. Gel-Filled Holders For Ultrasonic Transducers

    NASA Technical Reports Server (NTRS)

    Companion, John A.

    1992-01-01

    In new technique, ultrasonic transducer embedded in rubbery, castable, low-loss gel to enable transducer to "look" into surface of test object or human body at any desired angle. Composed of solution of water and ethylene glycol in collagen matrix. Provides total contact of water bath, also used on bodies or objects too large for water baths, even if moving. Also provides look angles of poly(methyl methacrylate) angle block with potential of reduced acoustic impedance and refraction. Custom-tailored to task at hand, and gel sufficiently inexpensive to be discarded upon completion. Easy to couple ultrasound in and out of gel, minimizing losses and artifacts of other types of standoffs employed in ultrasonic testing.

  6. Gel mobilities of linking-number topoisomers and their dependence on DNA helical repeat and elasticity

    PubMed Central

    Vetcher, Alexandre A.; McEwen, Abbye E.; Abujarour, Ramzey; Hanke, Andreas; Levene, Stephen D.

    2010-01-01

    Agarose-gel electrophoresis has been used for more than thirty years to characterize the linking-number (Lk) distribution of closed-circular DNA molecules. Although the physical basis of this technique remains poorly understood, the gel-electrophoretic behavior of covalently closed DNAs has been used to determine the local unwinding of DNA by proteins and small-molecule ligands, characterize supercoiling-dependent conformational transitions in duplex DNA, and to measure helical-repeat changes due to shifts in temperature and ionic strength. Those results have been analyzed by assuming that the absolute mobility of a particular topoisomer is mainly a function of the integral number of superhelical turns, and thus a slowly varying function of plasmid molecular weight. In examining the mobilities of Lk topoisomers for a series of plasmids that differ incrementally in size over more than one helical turn, we found that the size-dependent agarose-gel mobility of individual topoisomers with identical values of Lk (but different values of the excess linking number, ΔLk) vary dramatically over a duplex turn. Our results suggest that a simple semi-empirical relationship holds between the electrophoretic mobility of linking-number topoisomers and their average writhe in solution. PMID:20346570

  7. Investigations on gel forming media for use in low gravity bioseparations research

    NASA Astrophysics Data System (ADS)

    Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine; Kirkpatrick, Francis H.; Pike, Roland G.

    Microgravity research includes investigations designed to gain insight on methods of separating living cells. During a typical separation certain real-time measurements can be made by optical methods, but some materials must also be subjected to subsequent analyses, sometimes including cultivation of the separated cells. In the absence of on-orbit analytical or fraction collecting procedures, some means is required to ``capture'' cells after separation. The use of solutions that form gels was therefore investigated as a means of maintaining cells and/or macromolecules in the separated state after two types of simple ground-based experiments. Microgravity electrophoresis experiments were simulated by separating model cell types (rat, chicken, human and rabbit erythrocytes) in a vertical density gradient containing low-conductivity buffer, 1.7%-6.5% Ficoll, 6.8-5.0% sucrose, and 1% SeaPrep low-melting temperature agarose and demonstrating that, upon cooling, a gel formed in the column, and cells could be captured in the positions to which they had migrated. Two-phase extraction experiments were simulated by choosing two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2%), maltodextrin (5-7%) and gelatin (5-20%).

  8. Epithelial and stromal developmental patterns in a novel substitute of the human skin generated with fibrin-agarose biomaterials.

    PubMed

    Carriel, Víctor; Garzón, Ingrid; Jiménez, Jose-María; Oliveira, Ana-Celeste-Ximenes; Arias-Santiago, Salvador; Campos, Antonio; Sánchez-Quevedo, Maria-Carmen; Alaminos, Miguel

    2012-01-01

    Development of human skin substitutes by tissue engineering may offer new therapeutic alternatives to the use of autologous tissue grafts. For that reason, it is necessary to investigate and develop new biocompatible biomaterials that support the generation of a proper human skin construct. In this study, we generated a novel model of bioengineered human skin substitute using human cells obtained from skin biopsies and fibrin-agarose biomaterials and we evaluated this model both at the ex vivo and the in vivo levels. Once the dermal fibroblasts and the epithelial keratinocytes were isolated and expanded in culture, we used fibrin-agarose scaffolds for the development of a full-thickness human skin construct, which was evaluated after 1, 2, 3 and 4 weeks of development ex vivo. The skin substitutes were then grafted onto immune-deficient nude mice and analyzed at days 10, 20, 30 and 40 postimplantation using transmission electron microscopy, histochemistry and immunofluorescence. The results demonstrated that the fibrin-agarose artificial skin had adequate biocompatibility and proper biomechanical properties. A proper development of both the bioengineered dermis and epidermis was found after 30 days in vivo, although the tissues kept ex vivo and those implanted in the animal model for 10 or 20 days showed lower levels of differentiation. In summary, our model of fibrin-agarose skin equivalent was able to reproduce the structure and histological architecture of the native human skin, especially after long-term in vivo implantation, suggesting that these tissues could reproduce the native skin.

  9. No evidence of viral transmission following long-term implantation of agarose encapsulated porcine islets in diabetic dogs.

    PubMed

    Gazda, Lawrence S; Vinerean, Horatiu V; Laramore, Melissa A; Hall, Richard D; Carraway, Joseph W; Smith, Barry H

    2014-01-01

    We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n = 3) was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652) when compared to a first macrobead implantation (days 9, 21, and 21) in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years) implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

  10. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2004-03-01

    This technical progress report describes work performed from September 1, 2003, through February 29, 2004, for the project, ''Conformance Improvement Using Gels.'' We examined the properties of several ''partially formed'' gels that were formulated with a combination of high and low molecular weight HPAM polymers. After placement in 4-mm-wide fractures, these gels required about 25 psi/ft for brine to breach the gel (the best performance to date in fractures this wide). After this breach, stabilized residual resistance factors decreased significantly with increased flow rate. Also, residual resistance factors were up to 9 times greater for water than for oil. Nevertheless, permeability reduction factors were substantial for both water and oil flow. Gel with 2.5% chopped fiberglass effectively plugged 4-mm-wide fractures if a 0.5-mm-wide constriction was present. The ability to screen-out at a constriction appears crucial for particulate incorporation to be useful in plugging fractures. In addition to fiberglass, we examined incorporation of polypropylene fibers into gels. Once dispersed in brine or gelant, the polypropylene fibers exhibited the least gravity segregation of any particulate that we have tested to date. In fractures with widths of at least 2 mm, 24-hr-old gels (0.5% high molecular weight HPAM) with 0.5% fiber did not exhibit progressive plugging during placement and showed extrusion pressure gradients similar to those of gels without the fiber. The presence of the fiber roughly doubled the gel's resistance to first breach by brine flow. The breaching pressure gradients were not as large as for gels made with high and low molecular weight polymers (mentioned above). However, their material requirements and costs (i.e., polymer and/or particulate concentrations) were substantially lower than for those gels. A partially formed gel made with 0.5% HPAM did not enter a 0.052-mm-wide fracture when applying a pressure gradient of 65 psi/ft. This result

  11. Ring-Resonator/Sol-Gel Interferometric Immunosensor

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory; Cohen, David

    2007-01-01

    A proposed biosensing system would be based on a combination of (1) a sensing volume containing antibodies immobilized in a sol-gel matrix and (2) an optical interferometer having a ring resonator configuration. The antibodies would be specific to an antigen species that one seeks to detect. In the ring resonator of the proposed system, light would make multiple passes through the sensing volume, affording greater interaction length and, hence, greater antibody- detection sensitivity.

  12. Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells.

    PubMed

    Wlodek, D; Banáth, J; Olive, P L

    1991-11-01

    Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

  13. An extra peptide within the catalytic module of a β-agarase affects the agarose degradation pattern.

    PubMed

    Han, Wen-Jun; Gu, Jing-Yan; Liu, Hui-Hui; Li, Fu-Chuan; Wu, Zhi-Hong; Li, Yue-Zhong

    2013-03-29

    Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

  14. Behaviour of whey protein emulsion gel during oral and gastric digestion: effect of droplet size.

    PubMed

    Guo, Qing; Ye, Aiqian; Lad, Mita; Dalgleish, Douglas; Singh, Harjinder

    2014-06-21

    A set of whey protein stabilized-emulsion gels with different droplet size distributions (D4,3 = ∼1, 6 and 12 μm) was produced, and the mechanical properties of the gels in the linear viscoelastic region and at large deformation were measured, along with the physicochemical and structural changes of the gels during oral mastication and gastric digestion. The gels containing 1 μm oil droplets had an aggregated particle structure with proteins coating at oil droplets whereas the gels containing 12 μm oil droplets had a particle-filled structure with spatially continuous matrix. During oral processing, the release of oil droplets from the gels increased as the droplet size increased, with coalescence being seen in gels containing oil droplets of 6 and 12 μm diameter. Under gastric digestion, high degrees of coalescence and phase separation of oil droplets occurred in the gels containing 6 and 12 μm oil droplets because of oil droplet release from the gel matrix; this led to slow gastric emptying. The gels were finally broken down into peptide aggregates and oil droplets (or free oil). The gels, containing 1 μm oil droplets disintegrated into various particles of several to several tens of microns with a low degree of oil droplet release and coalescence. Protein breakdown was slower in these gels, suggesting that the protein structures of the gel matrices were affected by the sizes of the incorporated oil droplets.

  15. High-frequency alternating-crossed-field gel electrophoresis with neutral or slightly charged interpenetrating networks to improve DNA separation.

    PubMed

    Boyd, B M; Prausnitz, J M; Blanch, H W

    1998-12-01

    Toward improving DNA separations, this work reports the effects of high-frequency square-wave AC fields superimposed perpendicular to the direct current (DC) separation field on DNA migration in both polyacrylamide-based interpenetrating networks (IPNs) and in agarose networks. Compared to standard polyacrylamide gels, IPNs allow the separation of larger DNA (9000 bp vs. 5000 bp at 5 V/cm). In novel polyacrylamide-based IPNs, an alternating current (AC) field of 5 Hz increased the maximum DNA size separable. This effect was extended to larger DNA sizes with increasing electric-field strength up to and apparently beyond the power supply-limited maximum electric-field strength of 48 V/cm. The orthogonal AC field also increased mobility. These two results combine to yield a reduction in separation time of up to a factor of 20 in novel polyacrylamide-based IPNs. When negatively charged acrylic-acid groups were incorporated into the IPNs, the use of the AC field changed the DNA-network interaction, which altered the size dependence of DNA mobility. In agarose gels, an AC field of 50 Hz increased the size range separable; however, there was no increase in DNA mobility. There was no change in size dependence of mobility in an AC field when the number of charged groups in the agarose network was increased. Based on results in the literature, possible mechanisms were examined for the effects of the AC field on DNA separation.

  16. High-Frequency Alternating-Crossed-Field Gel Electrophoresis WithNeutral or Slightly Charged Interpenetrating Networks to Improve DNASeparation

    SciTech Connect

    Boyd, B.; Prausnitz, J.; Blanch, H.

    1998-07-01

    Toward improving DNA separations, this work reports theeffects of high-frequency square-wave AC fields superimposedperpendicular to the direct current (DC) separation field on DNAmigration in both polyacrylamide-based interpenetrating networks (IPNs)and in agarose networks. Compared to standard polyacrylamide gels, IPNsallow the separation of larger DNA (9000 bp vs. 5000 bp at 5 V/cm). Innovel polyacrylamide-based IPNs, an alternating current (AC) field of 5Hz increased the maximum DNA size separable. This effect was extended tolarger DNA sizes with increasing electric-field strength up to andapparently beyond the power supply-limited maximum electric-fieldstrength of 48 V/cm. The orthogonal AC field also increased mobility.These two results combine to yield a reduction in separation time of upto a factor of 20 in novel polyacrylamide-based IPNs. When negativelycharged acrylic-acid groups were incorporated into the IPNs, the use ofthe AC field changed the DNA-network interaction, which altered the sizedependence of DNA mobility. In agarose gels, an AC field of 50 Hzincreased the size range separable; however, there was no increase in DNAmobility. There was no change in size dependence of mobility in an ACfield when the number of charged groups in the agarose network wasincreased. Based on results in the literature, possible mechanisms wereexamined for the effects of the AC field on DNA separation.

  17. Drying SDS-Polyacrylamide Gels.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-09-01

    INTRODUCTIONThis protocol describes a method for drying SDS-polyacrylamide gels. Gels containing proteins radiolabeled with (35)S-labeled amino acids must be dried before autoradiographic images can be obtained. Nonradioactive gels can also be preserved by drying.

  18. Gel trapping of dense colloids.

    PubMed

    Laxton, Peter B; Berg, John C

    2005-05-01

    Phase density differences in sols, foams, or emulsions often lead to sedimentation or creaming, causing problems for materials where spatial uniformity over extended periods of time is essential. The problem may be addressed through the use of rheology modifiers in the continuous phase. Weak polymer gels have found use for this purpose in the food industry where they appear to be capable of trapping dispersoid particles in a three-dimensional matrix while displaying water-like viscosities at low shear. Attempts to predict sedimentation stability in terms of particle properties (size, shape, density difference) and gel yield stress have led to qualitative success for suspensions of large particles. The effect of particle size, however, in particular the case in which colloidal dimensions are approached, has not been investigated. The present work seeks to determine useful stability criteria for colloidal dispersions in terms of readily accessible viscoelastic descriptors. Results are reported for systems consisting of 12 microm poly(methyl methacrylate) (PMMA) spheres dispersed in aqueous gellan gum. Monovalent salt concentration is varied to control rheological properties, and sedimentation/centrifugation experiments are performed to determine dispersion stability. Necessary conditions for stability consist of a minimum yield stress together with a value of tan delta less than unity.

  19. An alternative easy method for antibody purification and analysis of protein-protein interaction using GST fusion proteins immobilized onto glutathione-agarose.

    PubMed

    Zalazar, L; Alonso, C A I; De Castro, R E; Cesari, A

    2014-01-01

    Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

  20. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    PubMed

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W

    2010-12-01

    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  1. Interfacing solid-state nanopores with gel media to slow DNA translocations.

    PubMed

    Waugh, Matthew; Carlsen, Autumn; Sean, David; Slater, Gary W; Briggs, Kyle; Kwok, Harold; Tabard-Cossa, Vincent

    2015-08-01

    We demonstrate the ability to slow DNA translocations through solid-state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores-presently one of the largest hurdles facing nanopore-based analysis-without affecting the signal quality or capture efficiency.

  2. New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase

    PubMed Central

    Cecchini, Davide A; Serra, Immacolata; Ubiali, Daniela; Terreni, Marco; Albertini, Alessandra M

    2007-01-01

    Background Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. Results Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. Conclusion The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues

  3. Fricke Gel Dosimeter Tissue-Equivalence a Monte Carlo Study

    NASA Astrophysics Data System (ADS)

    Valente, M.; Bartesaghi, G.; Gambarini, G.; Brusa, D.; Castellano, G.; Carrara, M.

    2008-06-01

    Gel dosimetry has proved to be a valuable technique for absorbed dose distribution measurements in radiotherapy. FriXy-gel dosimeters consist of Fricke (ferrous sulphate) solution infused with xylenol orange. The solution is incorporated to a gel matrix in order to fix it to a solid structure allowing good spatial resolution and is imaged with a transportable optical system, measuring visible light transmittance before and after irradiation. This paper presents an evaluation of total photon mass attenuation coefficients at energies in the range of 50 keV-10MeV for the radiochromic FriXy gel dosimeter sensitive material. Mass attenuation coefficient estimations have been performed by means of Monte Carlo (PENELOPE) simulations. These calculations have been carried out for the FriXy gel sensitive material as well as for soft tissue (ICRU) and pure liquid water; a comparison of the obtained data shows good agreement between the different materials.

  4. Conformance Improvement Using Gels

    SciTech Connect

    Seright, Randall S.; Schrader, Richard; II Hagstrom, John; Wang, Ying; Al-Dahfeeri, Abdullah; Gary, Raven; Marin; Amaury; Lindquist, Brent

    2002-09-26

    This research project had two objectives. The first objective was to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective was to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil.

  5. Crystallization from Gels

    NASA Astrophysics Data System (ADS)

    Narayana Kalkura, S.; Natarajan, Subramanian

    Among the various crystallization techniques, crystallization in gels has found wide applications in the fields of biomineralization and macromolecular crystallization in addition to crystallizing materials having nonlinear optical, ferroelectric, ferromagnetic, and other properties. Furthermore, by using this method it is possible to grow single crystals with very high perfection that are difficult to grow by other techniques. The gel method of crystallization provides an ideal technique to study crystal deposition diseases, which could lead to better understanding of their etiology. This chapter focuses on crystallization in gels of compounds that are responsible for crystal deposition diseases. The introduction is followed by a description of the various gels used, the mechanism of gelling, and the fascinating phenomenon of Liesegang ring formation, along with various gel growth techniques. The importance and scope of study on crystal deposition diseases and the need for crystal growth experiments using gel media are stressed. The various crystal deposition diseases, viz. (1) urolithiasis, (2) gout or arthritis, (3) cholelithiasis and atherosclerosis, and (4) pancreatitis and details regarding the constituents of the crystal deposits responsible for the pathological mineralization are discussed. Brief accounts of the theories of the formation of urinary stones and gallstones and the role of trace elements in urinary stone formation are also given. The crystallization in gels of (1) the urinary stone constituents, viz. calcium oxalate, calcium phosphates, uric acid, cystine, etc., (2) the constituents of the gallstones, viz. cholesterol, calcium carbonate, etc., (3) the major constituent of the pancreatic calculi, viz., calcium carbonate, and (4) cholic acid, a steroidal hormone are presented. The effect of various organic and inorganic ions, trace elements, and extracts from cereals, herbs, and fruits on the crystallization of major urinary stone and gallstone

  6. Cracks and Topological Defects in Lyotropic Nematic Gels

    NASA Astrophysics Data System (ADS)

    Islam, M. F.; Nobili, M.; Ye, Fangfu; Lubensky, T. C.; Yodh, A. G.

    2005-09-01

    We report on the effects of the coupling of nematic order and elasticity in anisotropic lyotropic gels consisting of large nematic domains of surfactant coated single wall carbon nanotubes embedded in a cross-linked N-isopropyl acrylamide polymer matrix. We observe the following striking features: (i) undulations and then cusping of the gel sidewalls, (ii) a nematic director field that evolves as the gel sidewalls deform, (iii) networks of surface cracks that are orthogonal to the nematic director field, and (iv) fissures at the sidewall cusps and associated topological defects that would not form in liquid nematics.

  7. In vivo remineralization of dentin using an agarose hydrogel biomimetic mineralization system

    NASA Astrophysics Data System (ADS)

    Han, Min; Li, Quan-Li; Cao, Ying; Fang, Hui; Xia, Rong; Zhang, Zhi-Hong

    2017-02-01

    A novel agarose hydrogel biomimetic mineralization system loaded with calcium and phosphate was used to remineralize dentin and induce the oriented densely parallel packed HA layer on defective dentin surface in vivo in a rabbit model. Firstly, the enamel of the labial surface of rabbits’ incisor was removed and the dentin was exposed to oral environment. Secondly, the hydrogel biomimetic mineralization system was applied to the exposed dentin surface by using a custom tray. Finally, the teeth were extracted and evaluated by scanning electron microscopy, X-ray diffraction, and nanoindentation test after a certain time of mineralization intervals. The regenerated tissue on the dentin surface was composed of highly organised HA crystals. Densely packed along the c axis, these newly precipitated HA crystals were perpendicular to the underlying dental surface with a tight bond. The demineralized dentin was remineralized and dentinal tubules were occluded by the grown HA crystals. The nanohardness and elastic modulus of the regenerated tissue were similar to natural dentin. The results indicated a potential clinical use for repairing dentin-exposed related diseases, such as erosion, wear, and dentin hypersensitivity.

  8. In vivo remineralization of dentin using an agarose hydrogel biomimetic mineralization system

    PubMed Central

    Han, Min; Li, Quan-Li; Cao, Ying; Fang, Hui; Xia, Rong; Zhang, Zhi-Hong

    2017-01-01

    A novel agarose hydrogel biomimetic mineralization system loaded with calcium and phosphate was used to remineralize dentin and induce the oriented densely parallel packed HA layer on defective dentin surface in vivo in a rabbit model. Firstly, the enamel of the labial surface of rabbits’ incisor was removed and the dentin was exposed to oral environment. Secondly, the hydrogel biomimetic mineralization system was applied to the exposed dentin surface by using a custom tray. Finally, the teeth were extracted and evaluated by scanning electron microscopy, X-ray diffraction, and nanoindentation test after a certain time of mineralization intervals. The regenerated tissue on the dentin surface was composed of highly organised HA crystals. Densely packed along the c axis, these newly precipitated HA crystals were perpendicular to the underlying dental surface with a tight bond. The demineralized dentin was remineralized and dentinal tubules were occluded by the grown HA crystals. The nanohardness and elastic modulus of the regenerated tissue were similar to natural dentin. The results indicated a potential clinical use for repairing dentin-exposed related diseases, such as erosion, wear, and dentin hypersensitivity. PMID:28167823

  9. Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans.