Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

PubMed Central

Murphy, Sandra; Dowling, Paul; Ohlendieck, Kay

2016-01-01

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. PMID:28248237

Gel Electrophoresis on a Budget to Dye for

ERIC Educational Resources Information Center

Yu, Julie H.

2010-01-01

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

Oral chronic ethanol administration to rodents by agar gel diet.

PubMed

Bykov, I; Palmén, M; Piirainen, L; Lindros, K O

2004-01-01

Chronic ethanol administration to rodents requires specially designed equipment and is labor intensive. Here we report a new procedure. A commercial liquid diet preparation was made into a gel by addition of 0.5% agar. The gel, containing 5.3% ethanol, was offered in Falcon tubes equipped with a feeding opening. The gel consumption by C57/Bl mice resulted in high blood ethanol levels (average 43 mM). After 6 weeks, marked liver steatosis and significantly increased serum alanine aminotransferase levels had developed. Administration of ethanol in a nutritionally adequate gel provides a simple method for studies on chronic ethanol effects in rodents.

Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

PubMed

Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

2013-09-15

Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of shape and size of agar gels on heating uniformity during pulsed microwave treatment.

PubMed

Soto-Reyes, Nohemí; Temis-Pérez, Ana L; López-Malo, Aurelio; Rojas-Laguna, Roberto; Sosa-Morales, María Elena

2015-05-01

Model gel systems with different shape (sphere, cylinder, and slab) and size (180 and 290 g) were prepared with agar (5%) and sucrose (5%). Dielectric constant (ε'), loss factor (ε"), thermophysical properties, and temperature distribution of the model system were measured. Each agar model system was immersed and suspended in water, and then, heated in a microwave oven with intermittent heating until the core temperature reached 50 °C. The ε' and ε" of agar gels decreased when frequency increased. The density and thermal conductivity values of the agar gels were 1033 kg/m(3) and 0.55 W/m °C, respectively. The temperature distribution of sphere, cylinder, and slab was different when similar power doses were applied. The slab reached 50 °C in less time (10 min) and showed a more uniform heating than spheres and cylinders in both sizes. Agar model systems of 180 g heated faster than those of 290 g. The coldest point was the center of the model systems in all studied cases. Shape and size are critical food factors that affect the heating uniformity during microwave heating processes. © 2015 Institute of Food Technologists®

Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

ERIC Educational Resources Information Center

Browning, Mark; Vanable, Joseph

2002-01-01

Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

Gel Electrophoresis of Gold-DNA Nanoconjugates

DOE PAGES

Pellegrino, T.; Sperling, R. A.; Alivisatos, A. P.; ...

2007-01-01

Gold-DNA conjugates were investigated in detail by a comprehensive gel electrophoresis study based on 1200 gels. A controlled number of single-stranded DNA of different length was attached specifically via thiol-Au bonds to phosphine-stabilized colloidal gold nanoparticles. Alternatively, the surface of the gold particles was saturated with single stranded DNA of different length either specifically via thiol-Au bonds or by nonspecific adsorption. From the experimentally determined electrophoretic mobilities, estimates for the effective diameters of the gold-DNA conjugates were derived by applying two different data treatment approaches. The first method is based on making a calibration curve for the relation between effectivemore » diameters and mobilities with gold nanoparticles of known diameter. The second method is based on Ferguson analysis which uses gold nanoparticles of known diameter as reference database. Our study shows that effective diameters derived from gel electrophoresis measurements are affected with a high error bar as the determined values strongly depend on the method of evaluation, though relative changes in size upon binding of molecules can be detected with high precision. Furthermore, in this study, the specific attachment of DNA via gold-thiol bonds to Au nanoparticles is compared to nonspecific adsorption of DNA. Also, the maximum number of DNA molecules that can be bound per particle was determined.« less

In-gel staining of proteins in native polyacrylamide gel electrophoresis using meso-tetrakis(4-sulfonatophenyl) porphyrin.

PubMed

Divakar, K; Devi, G Nandhini; Gautam, Pennathur

2012-01-01

Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining, and destaining of the gel, which are time-consuming and cumbersome. A new method for direct visualization of protein bands in PAGE has been developed using meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a dye without the need for any post-electrophoretic steps; thus, separation and recovery of enzymes become much easier for further analysis. Activity staining was carried out to show that the biochemical activity of the enzymes was preserved after electrophoresis.

Pump-probe imaging of nanosecond laser-induced bubbles in agar gel.

PubMed

Evans, R; Camacho-López, S; Pérez-Gutiérrez, F G; Aguilar, G

2008-05-12

In this paper we show results of Nd:YAG laser-induced bubbles formed in a one millimeter thick agar gel slab. The nine nanosecond duration pulse with a wave length of 532 nm was tightly focused inside the bulk of the gel sample. We present for the first time a pump-probe laser-flash shadowgraphy system that uses two electronically delayed Nd:YAG lasers to image the the bubble formation and shock wave fronts with nanosecond temporal resolution and up to nine seconds of temporal range. The shock waves generated by the laser are shown to begin at an earlier times within the laser pulse as the pulse energy increases. The shock wave velocity is used to infer a shocked to unshocked material pressure difference of up to 500 MPa. The bubble created settles to a quasi-stable size that has a linear relation to the maximum bubble size. The energy stored in the bubble is shown to increase nonlinearly with applied laser energy, and corresponds in form to the energy transmission in the agar gel. We show that the interaction is highly nonlinear, and most likely is plasma-mediated.

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.

PubMed

Wittig, Ilka; Karas, Michael; Schägger, Hermann

2007-07-01

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Starch gel electrophoresis of conifer seeds: a laboratory manual

Treesearch

M. Thompson Conkle; Paul D. Hodgskiss; Lucy B. Nunnally; Serena C. Hunter

1982-01-01

This manual describes fast, low-cost biochemical procedures for separating enzymes representing numerous genes of forest trees. During electrophoresis the mixture of enzymes from a megagametophyte or embryo of a germinated seed separates in a gel. Specific stains applied to gel slices locate each enzyme. These procedures expand on those developed for crops research....

Optimization of the Agar-gel Method for Isolation of Migrating Ascaris suum Larvae From the Liver and Lungs of Pigs

PubMed Central

Saeed, I; Roepstorff, A; Rasmussen, T; Høg, M; Jungersen, G

2001-01-01

Experiments on use of an agar-gel method for recovery of migrating Ascaris suum larvae from the liver and lungs of pigs were conducted to obtain fast standardized methods. Subsamples of blended tissues of pig liver and lungs were mixed with agar to a final concentration of 1% agar and the larvae allowed to migrate out of the agar-gel into 0.9% NaCl at 38°C. The results showed that within 3 h more than 88% of the recoverable larvae migrated out of the liver agar-gel and more than 83% of the obtained larvae migrated out of the lung agar-gel. The larvae were subsequently available in a very clean suspension which reduced the sample counting time. Blending the liver for 60 sec in a commercial blender showed significantly higher larvae recovery than blending for 30 sec. Addition of gentamycin to reduce bacterial growth during incubation, glucose to increase larval motility during migration or ice to increase sedimentation of migrated larvae did not influence larvae recovery significantly. PMID:11503373

Analyzing genetic diversity in conifers...isozyme resolution by starch gel electrophoresis

Treesearch

M. Thompson Conkle

1972-01-01

Enzymes in forest tree materials can be resolved by starch gel electrophoresis. A gel slab is prepared in a mold assembled from glass and plastic. Wicks containing an aqueous extract of macerated plant material are inserted in the gel and processed. The gel is sliced, stained, examined, and photographed. Isozyme bands produced by differential migration of enzymes...

Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels.

PubMed

Rehbein, Peter; Schwalbe, Harald

2015-06-01

Quantitative analysis of electrophoresis gels is an important part in molecular cloning, as well as in protein expression and purification. Parallel quantifications in yield and purity can be most conveniently obtained from densitometric analysis. This communication reports a comprehensive, reliable and simple protocol for gel quantification and documentation, applicable for single samples and with special features for protein expression screens. As major component of the protocol, the fully annotated code of a proprietary open source computer program for semi-automatic densitometric quantification of digitized electrophoresis gels is disclosed. The program ("GelQuant") is implemented for the C-based macro-language of the widespread integrated development environment of IGOR Pro. Copyright © 2014 Elsevier Inc. All rights reserved.

Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.

PubMed

Duncombe, Todd A; Herr, Amy E

2013-06-07

Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed. The fsPAG devices (120 μm tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast <1 h design-prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (<1 min) and short (1 mm) protein separations. The facile fabrication and prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

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.

Automatic DNA Diagnosis for 1D Gel Electrophoresis Images using Bio-image Processing Technique.

PubMed

Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Shaw, Philip J; Ukosakit, Kittipat; Tragoonrung, Somvong; Tongsima, Sissades

2015-01-01

DNA gel electrophoresis is a molecular biology technique for separating different sizes of DNA fragments. Applications of DNA gel electrophoresis include DNA fingerprinting (genetic diagnosis), size estimation of DNA, and DNA separation for Southern blotting. Accurate interpretation of DNA banding patterns from electrophoretic images can be laborious and error prone when a large number of bands are interrogated manually. Although many bio-imaging techniques have been proposed, none of them can fully automate the typing of DNA owing to the complexities of migration patterns typically obtained. We developed an image-processing tool that automatically calls genotypes from DNA gel electrophoresis images. The image processing workflow comprises three main steps: 1) lane segmentation, 2) extraction of DNA bands and 3) band genotyping classification. The tool was originally intended to facilitate large-scale genotyping analysis of sugarcane cultivars. We tested the proposed tool on 10 gel images (433 cultivars) obtained from polyacrylamide gel electrophoresis (PAGE) of PCR amplicons for detecting intron length polymorphisms (ILP) on one locus of the sugarcanes. These gel images demonstrated many challenges in automated lane/band segmentation in image processing including lane distortion, band deformity, high degree of noise in the background, and bands that are very close together (doublets). Using the proposed bio-imaging workflow, lanes and DNA bands contained within are properly segmented, even for adjacent bands with aberrant migration that cannot be separated by conventional techniques. The software, called GELect, automatically performs genotype calling on each lane by comparing with an all-banding reference, which was created by clustering the existing bands into the non-redundant set of reference bands. The automated genotype calling results were verified by independent manual typing by molecular biologists. This work presents an automated genotyping tool from DNA

Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.

PubMed

Lohnes, Karen; Quebbemann, Neil R; Liu, Kate; Kobzeff, Fred; Loo, Joseph A; Ogorzalek Loo, Rachel R

2016-07-15

The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel.

PubMed

Slotved, H C; Barnes, E H; Bjørn, H; Christensen, C M; Eriksen, L; Roepstorff, A; Nansen, P

1996-06-01

Four groups with three pigs in each group were inoculated with Oesophagostomum dentatum larvae (L3 larvae). Groups 1 and 3 were inoculated with 20,000 larvae, and Groups 2 and 4 with 200,000 larvae. On Days 11 and 34, respectively, Groups 1 and 2 and Groups 3 and 4 were slaughtered, and the contents from the large intestines collected. Subsamples of intestinal contents were mixed with agar to a final concentration of 1% agar and allowed to set. The worms were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight. Then the worms were collected on a sieve (38 microns mesh) and counted. The worms retained in the agar-gel were counted after pouring the melted agar through a sieve (38 microns mesh). The results showed that more than 95% of the worms migrated out of the agar-gel, and subsequently were available for counting in an almost clean suspension. Additionally the method yielded a high worm recovery; all stages were recovered. The recovery percentage was not significantly affected by either the dose of parasites or the time interval from slaughtering to start of incubation (37-128 min).

Increase in local protein concentration by field-inversion gel electrophoresis.

PubMed

Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

2007-09-26

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

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

PubMed Central

Stellwagen, Nancy C.

2009-01-01

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 due primarily 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) × 10-4 cm2/Vs in 40 mM Tris-acetate-EDTA buffer at 20°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. PMID:19517510

Disposable pen-shaped capillary gel electrophoresis cartridge for fluorescence detection of bio-molecules

NASA Astrophysics Data System (ADS)

Amirkhanian, Varoujan; Tsai, Shou-Kuan

2014-03-01

We introduce a novel and cost-effective capillary gel electrophoresis (CGE) system utilizing disposable pen-shaped gelcartridges for highly efficient, high speed, high throughput fluorescence detection of bio-molecules. The CGE system has been integrated with dual excitation and emission optical-fibers with micro-ball end design for fluorescence detection of bio-molecules separated and detected in a disposable pen-shaped capillary gel electrophoresis cartridge. The high-performance capillary gel electrophoresis (CGE) analyzer has been optimized for glycoprotein analysis type applications. Using commercially available labeling agent such as ANTS (8-aminonapthalene-1,3,6- trisulfonate) as an indicator, the capillary gel electrophoresis-based glycan analyzer provides high detection sensitivity and high resolving power in 2-5 minutes of separations. The system can hold total of 96 samples, which can be automatically analyzed within 4-5 hours. This affordable fiber optic based fluorescence detection system provides fast run times (4 minutes vs. 20 minutes with other CE systems), provides improved peak resolution, good linear dynamic range and reproducible migration times, that can be used in laboratories for high speed glycan (N-glycan) profiling applications. The CGE-based glycan analyzer will significantly increase the pace at which glycoprotein research is performed in the labs, saving hours of preparation time and assuring accurate, consistent and economical results.

Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

USDA-ARS?s Scientific Manuscript database

Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

PubMed

Flórez, Ana Belén; Mayo, Baltasar

2015-12-02

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Use of an agar-gel technique for large scale application to recover Ascaris suum larvae from intestinal contents of pigs.

PubMed

Slotved, H C; Barnes, E H; Eriksen, L; Roepstorff, A; Nansen, P; Bjørn, H

1997-01-01

Four groups each of 3 pigs were inoculated with Ascaris suum eggs. Pigs in groups 1 and 3 were inoculated with 1000 eggs, and pigs in groups 2 and 4 with 10,000 eggs. On day 10 and 21 post-inoculation (p.i.), respectively, groups 1 + 2 and 3 + 4 were slaughtered, and the contents from the small intestines collected. The contents were mixed with agar to a final concentration of 1% agar and allowed to sediment. The larvae were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight, and were then collected on a sieve (20 microns mesh) and counted. The larvae retained in the agar-gel were counted after pouring the melted agar through a sieve (20 microns mesh). The results showed that more than 97% of the larvae migrated out of the agar-gel and were available for counting in an almost clean suspension. The inoculation dose level did not significantly affect the recovery percentage, neither did the larval stage (10 or 21 days old larvae). The variation in the time interval from slaughtering to start of incubation (interval 57-155 min) did not significantly affect the recovery percentage.

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel.

PubMed

Nosal, P; Christensen, C M; Nansen, P

1998-01-01

The agar-gel migration technique has previously been described, however, aspects regarding the effect of timing on worm migration needed further scrutiny. In the first experiment, pigs inoculated with Oesophagostomum dentatum were slaughtered simultaneously and their intestines stored at 21-23 degrees C until processed pairwise 2, 4, 6, 8, 12 and 18 h after slaughter. More than 95% of the worms migrated out of the agar if processed within 6 h. In the second experiment, intestines were treated immediately after slaughter and the migratory speed of adult worms or 4th-stage larvae of O. dentatum or O. quadrispinulatum, or adult Hyostrongylus rubidus were studied. For both Oesophagostomum species, more than 90% of the worms were recovered within 1 h. H. rubidus was significantly slower; however, approximately 98% of the worms had migrated out of the agar-gel by 20 h. This information is essential in planning experiments where recovery of live worms is of value.

Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: application to agarose gel and a polystyrene particle.

PubMed

Tietz, D; Gombocz, E; Chrambach, A

1991-10-01

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle-free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0-20% 5-(N-2,3-dihydroxypropylacetamido)- 2,4,6-triiodo-N,N'bis-(2,3-dihydroxypropyl)-isophthalamide (Nycodenz). Densitometry of gelled-in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time-lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109-169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user-friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135-157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle-free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis.

PubMed

Riond, Barbara; Wenger-Riggenbach, Bettina; Hofmann-Lehmann, Regina; Lutz, Hans

2009-03-01

Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and alpha-, beta-, and gamma-globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm-blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi-automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within-run and within-assay precision. Data from warm-blooded and draught horses were compared using the Mann-Whitney U test. Within-run and within-assay CVs were <5% for all protein fractions. No significant difference was found between warm-blooded and heavy draught horses and so combined reference intervals (2.5-97.5%) were calculated for total protein (51.0-72.0 g/L), albumin (29.6-38.5 g/L), alpha(1)-globulin (1.9-3.1 g/L), alpha(2)-globulin (5.3-8.7 g/L), beta(1)-globulin (2.8-7.3g/L), beta(2)-globulin (2.2-6.0 g/L), and gamma-globulin (5.8-12.7 g/L) concentrations, and albumin/globulin ratio (0.93-1.65). Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.

Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

PubMed

Xia, Zhi-Jun; Liu, Zhen; Kong, Fan-Zhi; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

2017-12-01

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Simple and rapid system for two-dimensional gel electrophoresis technique: A laboratory exercise for high school students.

PubMed

Maurye, Praveen; Basu, Arpita; Biswas, Jayanta Kumar; Bandyopadhyay, Tapas Kumar; Naskar, Malay

2018-02-28

Polyacrylamide gel electrophoresis (PAGE) is the most classical technique favored worldwide for resolution of macromolecules in many biochemistry laboratories due to its incessant advanced developments and wide modifications. These ever-growing advancements in the basic laboratory equipments lead to emergence of many expensive, complex, and tricky laboratory equipments. Practical courses of biochemistry at high school or undergraduate levels are often affected by these complications. Two dimensional gel electrophoresis technique (2D-PAGE) used for resolving thousands of proteins in a gel is a combination of isoelectric focusing (first dimension gel electrophoresis technique) and sodium-dodecylsulphate PAGE (second dimension gel electrophoresis technique or SDS-PAGE). Two different laboratory equipments are needed to carry out effective 2D-PAGE technique, which also invites extra burden to the school laboratory. Here, we describe a low cost, time saving and simple gel cassette for protein 2D-PAGE technique that uses easily fabricated components and routine off-the-shelf materials. The performance of the apparatus was verified in a practical exercise by a group of high school students with positive outcomes. © 2018 by The International Union of Biochemistry and Molecular Biology, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.

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). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PFGE MAPPER and PFGE READER: two tools to aid in the analysis and data input of pulse field gel electrophoresis maps.

PubMed Central

Shifman, M. A.; Nadkarni, P.; Miller, P. L.

1992-01-01

Pulse field gel electrophoresis mapping is an important technique for characterizing large segments of DNA. We have developed two tools to aid in the construction of pulse field electrophoresis gel maps: PFGE READER which stores experimental conditions and calculates fragment sizes and PFGE MAPPER which constructs pulse field gel electrophoresis maps. PMID:1482898

Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

PubMed Central

Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

2008-01-01

Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments done in lab. Here we report the development and implementation of novel exercises that integrate the biological concepts of DNA structure and replication with the techniques of PCR and gel electrophoresis. Learning goals were defined based on concepts taught throughout the cell biology lab course and learning objectives specific to the PCR and gel electrophoresis lab. Exercises developed to promote critical thinking and target the underlying concepts of PCR, primer design, gel analysis, and troubleshooting were incorporated into an existing lab unit based on the detection of genetically modified organisms. Evaluative assessments for each exercise were aligned with the learning goals and used to measure student learning achievements. Our analysis found that the exercises were effective in enhancing student understanding of these concepts as shown by student performance across all learning goals. The new materials were particularly helpful in acquiring relevant knowledge, fostering critical-thinking skills, and uncovering prevalent misconceptions. PMID:18316813

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

PubMed

Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

2003-01-01

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Hybrid slab-microchannel gel electrophoresis system

DOEpatents

Balch, Joseph W.; Carrano, Anthony V.; Davidson, James C.; Koo, Jackson C.

1998-01-01

A hybrid slab-microchannel gel electrophoresis system. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

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.

Analysis of Soluble Proteins in Natural Cordyceps sinensis from Different Producing Areas by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Two-dimensional Electrophoresis.

PubMed

Li, Chun-Hong; Zuo, Hua-Li; Zhang, Qian; Wang, Feng-Qin; Hu, Yuan-Jia; Qian, Zheng-Ming; Li, Wen-Jia; Xia, Zhi-Ning; Yang, Feng-Qing

2017-01-01

As one of the bioactive components in Cordyceps sinensis (CS), proteins were rarely used as index components to study the correlation between the protein components and producing areas of natural CS. Protein components of 26 natural CS samples produced in Qinghai, Tibet, and Sichuan provinces were analyzed and compared to investigate the relationship among 26 different producing areas. Proteins from 26 different producing areas were extracted by Tris-HCl buffer with Triton X-100, and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). The SDS-PAGE results indicated that the number of protein bands and optical density curves of proteins in 26 CS samples was a bit different. However, the 2-DE results showed that the numbers and abundance of protein spots in protein profiles of 26 samples were obviously different and showed certain association with producing areas. Based on the expression values of matched protein spots, 26 batches of CS samples can be divided into two main categories (Tibet and Qinghai) by hierarchical cluster analysis. The number of protein bands and optical density curves of proteins in 26 Cordyceps sinensis samples were a bit different on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profilesNumbers and abundance of protein spots in protein profiles of 26 samples were obvious different on two-dimensional electrophoresis mapsTwenty-six different producing areas of natural Cordyceps sinensis samples were divided into two main categories (Tibet and Qinghai) by Hierarchical cluster analysis based on the values of matched protein spots. Abbreviations Used : SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, Cordyceps sinensis : CS, TCMs: Traditional Chinese medicines.

Hybrid slab-microchannel gel electrophoresis system

DOEpatents

Balch, J.W.; Carrano, A.V.; Davidson, J.C.; Koo, J.C.

1998-05-05

A hybrid slab-microchannel gel electrophoresis system is described. The hybrid system permits the fabrication of isolated microchannels for biomolecule separations without imposing the constraint of a totally sealed system. The hybrid system is reusable and ultimately much simpler and less costly to manufacture than a closed channel plate system. The hybrid system incorporates a microslab portion of the separation medium above the microchannels, thus at least substantially reducing the possibility of non-uniform field distribution and breakdown due to uncontrollable leakage. A microslab of the sieving matrix is built into the system by using plastic spacer materials and is used to uniformly couple the top plate with the bottom microchannel plate. 4 figs.

Discolored Red Seaweed Pyropia yezoensis with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides.

PubMed

Sasuga, Keiji; Yamanashi, Tomoya; Nakayama, Shigeru; Ono, Syuetsu; Mikami, Koji

2018-04-26

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.

System for loading slab-gel holders for electrophoresis separation

DOEpatents

Anderson, Norman G.; Anderson, Norman L.

1979-01-01

A slab-gel loading system includes a prismatic chamber for filling a plurality of slab-gel holders simultaneously. Each slab-gel holder comprises a pair of spaced apart plates defining an intermediate volume for gel containment. The holders are vertically positioned in the chamber with their major surfaces parallel to the chamber end walls. A liquid inlet is provided at the corner between the bottom and a side wall of the chamber for distributing a polymerizable monomer solution or a coagulable colloidal solution into each of the holders. The chamber is rotatably supported so that filling can begin with the corner having the liquid inlet directed downwardly such that the solution is gently funneled upwardly, without mixing, along the diverging side and bottom surfaces. As filling proceeds, the chamber is gradually rotated to position the bottom wall in a horizontal mode. The liquid filling means includes a plastic envelope with a septum dividing it into two compartments for intermixing two solutions of different density and thereby providing a liquid flow having a density gradient. The resulting gels have a density gradient between opposite edges for subsequent use in electrophoresis separations.

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

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments.

PubMed

Jaeger, Philipp A; McElfresh, Cameron; Wong, Lily R; Ideker, Trey

2015-08-15

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of Soluble Proteins in Natural Cordyceps sinensis from Different Producing Areas by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Two-dimensional Electrophoresis

PubMed Central

Li, Chun-Hong; Zuo, Hua-Li; Zhang, Qian; Wang, Feng-Qin; Hu, Yuan-Jia; Qian, Zheng-Ming; Li, Wen-Jia; Xia, Zhi-Ning; Yang, Feng-Qing

2017-01-01

Background: As one of the bioactive components in Cordyceps sinensis (CS), proteins were rarely used as index components to study the correlation between the protein components and producing areas of natural CS. Objective: Protein components of 26 natural CS samples produced in Qinghai, Tibet, and Sichuan provinces were analyzed and compared to investigate the relationship among 26 different producing areas. Materials and Methods: Proteins from 26 different producing areas were extracted by Tris-HCl buffer with Triton X-100, and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Results: The SDS-PAGE results indicated that the number of protein bands and optical density curves of proteins in 26 CS samples was a bit different. However, the 2-DE results showed that the numbers and abundance of protein spots in protein profiles of 26 samples were obviously different and showed certain association with producing areas. Conclusions: Based on the expression values of matched protein spots, 26 batches of CS samples can be divided into two main categories (Tibet and Qinghai) by hierarchical cluster analysis. SUMMARY The number of protein bands and optical density curves of proteins in 26 Cordyceps sinensis samples were a bit different on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profilesNumbers and abundance of protein spots in protein profiles of 26 samples were obvious different on two-dimensional electrophoresis mapsTwenty-six different producing areas of natural Cordyceps sinensis samples were divided into two main categories (Tibet and Qinghai) by Hierarchical cluster analysis based on the values of matched protein spots. Abbreviations Used: SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 2-DE: Two-dimensional electrophoresis, Cordyceps sinensis: CS, TCMs: Traditional Chinese medicines PMID:28250651

Electrophoresis gel image processing and analysis using the KODAK 1D software.

PubMed

Pizzonia, J

2001-06-01

The present article reports on the performance of the KODAK 1D Image Analysis Software for the acquisition of information from electrophoresis experiments and highlights the utility of several mathematical functions for subsequent image processing, analysis, and presentation. Digital images of Coomassie-stained polyacrylamide protein gels containing molecular weight standards and ethidium bromide stained agarose gels containing DNA mass standards are acquired using the KODAK Electrophoresis Documentation and Analysis System 290 (EDAS 290). The KODAK 1D software is used to optimize lane and band identification using features such as isomolecular weight lines. Mathematical functions for mass standard representation are presented, and two methods for estimation of unknown band mass are compared. Given the progressive transition of electrophoresis data acquisition and daily reporting in peer-reviewed journals to digital formats ranging from 8-bit systems such as EDAS 290 to more expensive 16-bit systems, the utility of algorithms such as Gaussian modeling, which can correct geometric aberrations such as clipping due to signal saturation common at lower bit depth levels, is discussed. Finally, image-processing tools that can facilitate image preparation for presentation are demonstrated.

Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support.

PubMed

Li, Tuoping; Li, Suhong; Wang, Na; Tain, Lirui

2008-08-15

Pectinase was immobilized on an activated agar-gel support by multipoint attachment. The maximal activity of immobilized pectinase was obtained at 5°C, pH 3.6, with a 24h reaction time at an enzyme dose of 0.52mg protein/g gel, and the gel was activated with 1.0M glycidol. These conditions increased the thermal stability of the immobilized pectinase 19-fold compared with the free enzyme at 65°C. The optimal temperature for pectinase activity changed from 40 to 50°C after immobilization; however, the optimal pH remained unchanged. The immobilized enzyme also exhibited great operational stability, and an 81% residual activity was observed in the immobilized enzyme after 10 batch reactions. Copyright © 2008 Elsevier Ltd. All rights reserved.

Enzymatic desulfation of the red seaweeds agar by Marinomonas arylsulfatase.

PubMed

Wang, Xueyan; Duan, Delin; Fu, Xiaoting

2016-12-01

Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from 13 C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

PubMed Central

Khan, Sharik R.; Kuzminov, Andrei

2013-01-01

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

PubMed

Luo, Xiaoting; Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

2017-02-02

Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Coupling Sodium Dodecyl Sulfate–Capillary Polyacrylamide Gel Electrophoresis with MALDI-TOF-MS via a PTFE Membrane

PubMed Central

Lu, Joann J.; Zhu, Zaifang; Wang, Wei; Liu, Shaorong

2011-01-01

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. PMID:21309548

Salmonella enterica Pulsed-Field Gel Electrophoresis Clusters, Minnesota, USA, 2001–2007

PubMed Central

Hedberg, Craig W.; Meyer, Stephanie; Boxrud, David J.; Smith, Kirk E.

2010-01-01

We determined characteristics of Salmonella enterica pulsed-field gel electrophoresis clusters that predict their being solved (i.e., that result in identification of a confirmed outbreak). Clusters were investigated by the Minnesota Department of Health by using a dynamic iterative model. During 2001–2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of >4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the first 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the first 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-field gel electrophoresis clusters, investigation of clusters of >4 cases and clusters in which the first 3 case isolates were received at a public health laboratory within 7 days may improve outbreak investigations. PMID:21029524

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis.

PubMed

Mineyama, R; Yoshino, S; Fukushima, K

2004-01-01

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

PubMed

Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

2017-03-01

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

NASA Astrophysics Data System (ADS)

Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

2009-11-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

PubMed Central

Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong

2013-01-01

In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl

2009-10-02

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this systemmore » using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.« less

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

PubMed

Newby, A C; Chrambach, A

1979-02-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

PubMed Central

Newby, Andrew C.; Chrambach, Andreas

1979-01-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

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…

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

PubMed Central

Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

2012-01-01

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

Sol-gel chemistry-based Ucon-coated columns for capillary electrophoresis.

PubMed

Hayes, J D; Malik, A

1997-07-18

A sol-gel chemistry-based novel approach for the preparation of a Ucon-coated fused-silica capillary column in capillary electrophoresis is presented. In this approach the sol-gel process is carried out inside 25 microm I.D. fused-silica capillaries. The sol solution contained appropriate quantities of an alkoxide-based sol-gel precursor, a polymeric coating material (Ucon), a crosslinking reagent, a surface derivatizing reagent, controlled amounts of water and a catalyst dissolved in a suitable solvent system. The coating procedure involves filling a capillary with the sol solution and allowing the sol-gel process to proceed for an optimum period. Hydrolysis of the alkoxide precursor and polycondensation of the hydrolyzed products with the surface silanol groups and the hydroxy-terminated Ucon molecules lead to the formation of a surface-bonded sol-gel coating on the inner walls of the capillary. The thickness of the coated film can be controlled by varying the reaction time, coating solution composition and experimental conditions. Commercial availability of high purity sol-gel precursors (e.g., TEOS 99.999%), the ease of coating, run-to-run and column-to-column reproducibility, and long column lifetimes make sol-gel coating chemistry very much suitable for being applied in analytical microseparations column technology. Test samples of basic proteins and nucleotides were used to evaluate the column performance. These results show that the sol-gel coating scheme has allowed for the generation of bio-compatible surfaces characterized by high separation efficiencies in CE. For different types of solutes, the sol-gel coated Ucon column consistently provided migration time R.S.D. values of the order of 0.5%.

Aligning Goals, Assessments, and Activities: An Approach to Teaching PCR and Gel Electrophoresis

ERIC Educational Resources Information Center

Phillips, Allison R.; Robertson, Amber L.; Batzli, Janet; Harris, Michelle; Miller, Sarah

2008-01-01

Polymerase chain reaction (PCR) and gel electrophoresis have become common techniques used in undergraduate molecular and cell biology labs. Although students enjoy learning these techniques, they often cannot fully comprehend and analyze the outcomes of their experiments because of a disconnect between concepts taught in lecture and experiments…

Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

PubMed

Santos-Sanches, Ilda; Chambel, Lélia; Tenreiro, Rogério

2015-01-01

Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

A Sol-Gel-Modified Poly(methyl methacrylate) Electrophoresis Microchip with a Hydrophilic Channel Wall

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Gang; Xu, Xuejiao; Lin, Yuehe

2007-07-27

A sol-gel method was employed to fabricate a poly(methyl methacrylate) (PMMA) electrophoresis microchip that contains a hydrophilic channel wall. To fabricate such a device, tetraethoxysilane (TEOS) was injected into the PMMA channel and was allowed to diffuse into the surface layer for 24 h. After removing the excess TEOS, the channel was filled with an acidic solution for 3 h. Subsequently, the channel was flushed with water and was pretreated in an oven to obtain a sol-gel-modified PMMA microchip. The water contact angle for the sol-gel-modified PMMA was 27.4° compared with 66.3° for the pure PMMA. In addition, the electro-osmoticmore » flow increased from 2.13×10-4 cm2 V-1 s-1 for the native-PMMA channel to 4.86×10-4 cm2 V-1 s-1 for the modified one. The analytical performance of the sol-gel-modified PMMA microchip was demonstrated for the electrophoretic separation of several purines, coupled with amperometric detection. The separation efficiency of uric acid increased to 74 882.3 m-1 compared with 14 730.5 m-1 for native-PMMA microchips. The result of this simple modification is a significant improvement in the performance of PMMA for microchip electrophoresis and microfluidic applications.« less

Optimization of Large Gel 2D Electrophoresis for Proteomic Studies of Skeletal Muscle

PubMed Central

Reed, Patrick W.; Densmore, Allison; Bloch, Robert J.

2013-01-01

We describe improved methods for large format, 2-dimensional gel electrophoresis (2-DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7M urea + 2 M thiourea, instead of 9M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2D-GE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes. PMID:22589104

On gel electrophoresis of dielectric charged particles with hydrophobic surface: A combined theoretical and numerical study.

PubMed

Majee, Partha Sarathi; Bhattacharyya, Somnath; Gopmandal, Partha Pratim; Ohshima, Hiroyuki

2018-03-01

A theoretical study on the gel electrophoresis of a charged particle incorporating the effects of dielectric polarization and surface hydrophobicity at the particle-liquid interface is made. A simplified model based on the weak applied field and low charge density assumption is also presented and compared with the full numerical model for a nonpolarizable particle to elucidate the nonlinear effects such as double layer polarization and relaxation as well as surface conduction. The main motivation of this study is to analyze the electrophoresis of the surface functionalized nanoparticle with tunable hydrophobicity or charged fluid drop in gel medium by considering the electrokinetic effects and hydrodynamic interactions between the particle and the gel medium. An effective medium approach, in which the transport in the electrolyte-saturated hydrogel medium is governed by the Brinkman equation, is adopted in the present analysis. The governing electrokinetic equations based on the conservation principles are solved numerically. The Navier-slip boundary condition along with the continuity condition of dielectric displacement are imposed on the surface of the hydrophobic polarizable particle. The impact of the slip length on the electrophoresis is profound for a thinner Debye layer, however, surface conduction effect also becomes significant for a hydrophobic particle. Impact of hydrophobicity and relaxation effects are higher for a larger particle. Dielectric polarization creates a reduction in its electrophoretic propulsion and has negligible impact at the thinner Debye length as well as lower gel screening length. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis.

PubMed

Li, Yuping; Deng, Dandan; Zhang, Xuebin; Zhang, Haina; Wang, Cong; Chen, Peng

2013-12-15

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds. Copyright © 2013 Elsevier Inc. All rights reserved.

Rapid pulsed-field gel electrophoresis protocol for subtyping of Streptococcus suis serotype 2.

PubMed

Luey, Cindy K Y; Chu, Yiu Wai; Cheung, Terence K M; Law, Catherine C P; Chu, Man Yu; Cheung, Danny T L; Kam, Kai Man

2007-03-01

A rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Streptococcus suis serotype 2 was developed and evaluated using 27 clinical isolates from 22 epidemiologically unrelated patients. Results were matched against antibiogram, virulence genotyping and multi locus sequence typing (MLST). PFGE appeared to be the most discriminatory with numerical index of discrimination (D) equal to 0.87.

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.

Informatics and Statistics for Analyzing 2-D Gel Electrophoresis Images

PubMed Central

Dowsey, Andrew W.; Morris, Jeffrey S.; Gutstein, Howard B.; Yang, Guang-Zhong

2013-01-01

Despite recent progress in “shotgun” peptide separation by integrated liquid chromatography and mass spectrometry (LC/MS), proteome coverage and reproducibility are still limited with this approach and obtaining enough replicate runs for biomarker discovery is a challenge. For these reasons, recent research demonstrates that there is a continuing need for protein separation by two-dimensional gel electrophoresis (2-DE). However, with traditional 2-DE informatics, the digitized images are reduced to symbolic data through spot detection and quantification before proteins are compared for differential expression by spot matching. Recently, a more robust and automated paradigm has emerged where gels are directly aligned in the image domain before spots are detected across the whole image set as a whole. In this chapter, we describe the methodology for both approaches and discuss the pitfalls present when reasoning statistically about the differential protein expression discovered. PMID:20013375

Erythrocyte membrane protein analysis by sodium dodecyl sulphate-capillary gel electrophoresis in the diagnosis of hereditary spherocytosis.

PubMed

Debaugnies, France; Cotton, Frédéric; Boutique, Charles; Gulbis, Béatrice

2011-03-01

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is currently the reference method for detecting protein deficiencies related to hereditary spherocytosis. The aim of the study was to evaluate an automated capillary gel electrophoresis system, the Experion instrument from BioRad, for its ability to separate and quantify the erythrocyte membrane proteins. The major erythrocyte membrane proteins (actin, protein 4.2, protein 4.1, band 3, ankyrin, α- and β-spectrin) were extracted and purified from membrane ghosts by centrifugation, immunoprecipitation and electroelution. Analyses were performed using SDS-PAGE and sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE) to establish a separation profile of the total ghosts. Then, the samples from patients received for investigations of erythrocyte membrane defects were analysed. Five of the seven expected erythrocyte membrane proteins were finally separated and identified. In the 20 studied cases, taking into account the screening test results and the clinical and family histories, the SDS-CGE method allowed us to achieve the same conclusion as with SDS-PAGE, except for the patient with elliptocytosis. The new SDS-CGE method presents interesting features that could make this instrument a powerful diagnostic tool for detection of erythrocyte membrane protein abnormalities, and can be proposed as an automated alternative method to the labour intensive SDS-PAGE analysis.

Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

PubMed Central

Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

2016-01-01

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

Experimenting with a Visible Copper-Aluminum Displacement Reaction in Agar Gel and Observing Copper Crystal Growth Patterns to Engage Student Interest and Inquiry

ERIC Educational Resources Information Center

Xu, Xinhua; Wu, Meifen; Wang, Xiaogang; Yang, Yangyiwei; Shi, Xiang; Wang, Guoping

2016-01-01

The reaction process of copper-aluminum displacement in agar gel was observed at the microscopic level with a stereomicroscope; pine-like branches of copper crystals growing from aluminum surface into gel at a constant rate were observed. Students were asked to make hypotheses on the pattern formation and design new research approaches to prove…

Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

PubMed

Hsieh, Ming-Kun; Shih, Pei-Yin; Wei, Chia-Fong; Vickroy, Thomas W; Chou, Chi-Chung

2016-03-30

The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

Characterization of a novel alkaline arylsulfatase from Marinomonas sp. FW-1 and its application in the desulfation of red seaweed agar.

PubMed

Wang, Xueyan; Duan, Delin; Xu, Jiachao; Gao, Xin; Fu, Xiaoting

2015-10-01

A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 μM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

PubMed

Hayakawa, Mitsuo; Hosogi, Yumiko; Takiguchi, Hisashi; Shiroza, Teruaki; Shibata, Yasuko; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Hamajima, Susumu; Abiko, Yoshimitsu

2003-02-01

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale. Copyright 2003 Elsevier Science (USA)

[Preparation of polyacrylamide gel electrophoresis for human chorionic gonadotropin chimeric peptide 12 expressed in E. coli].

PubMed

Zou, Yong-Shui; Xu, Wan-Xiang; He, Yuan; Sun, Zhi-Da; Xue, Xiao-Lin

2002-09-01

In recent years, development of chimeric peptide (CP) immunogens is a trend in the vaccinological field. The CPs contain a B cell epitope(s) of target antigen and a promiscuous self - or foreign- T cell epitope(s). However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To purify the human chorionic gonadotropin (hCG) CP12 expressed in E. coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel- carrying plate when the bromophenol blue band arrived at 1-1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer. About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone. Each sample was dissolved in 20 microL sample buffer and analyzed and identified by SDS-PAGE and Western blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50-100 microgram level after a single-step purification of this preparative PAGE, with respect to the sample which contained 3-4 mg of cell proteins.

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip.

PubMed

Tsai, Shuo-Wen; Loughran, Michael; Suzuki, Hiroaki; Karube, Isao

2004-02-01

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.

Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

PubMed

Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

2016-01-29

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Karyotype analysis of anuran trypanosomes by pulsed-field gradient gel electrophoresis.

PubMed

Lun, Z R; Desser, S S

1995-12-01

The chromosomes of 12 species and isolates of anuran trypanosomes were investigated by pulsed-field gradient gel electrophoresis. Twelve to 16 chromosomes ranging from 0.45 to 2.2 megabase pairs were found in each of these trypanosomes. Minichromosomes were not observed in any of the examined isolates. Results indicate that different species of anuran trypanosomes display distinct karyotype patterns, and that isolates from the same region are similar. Our findings also reveal that most chromosome profiles of these trypanosomes are in accordance with isoenzyme and random amplified polymorphic DNA analysis data.

Protein Separation by Capillary Gel Electrophoresis: A Review

PubMed Central

Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

2011-01-01

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

Light transfer in agar immobilized microalgae cell cultures

NASA Astrophysics Data System (ADS)

Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

2017-09-01

This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

PubMed

Nakano, Keiichi; Tamura, Shogo; Otuka, Kohei; Niizeki, Noriyasu; Shigemura, Masahiko; Shimizu, Chikara; Matsuno, Kazuhiko; Kobayashi, Seiichi; Moriyama, Takanori

2013-07-15

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

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). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

Two Electrophoresis Experiments for Freshmen in the Health Professions.

ERIC Educational Resources Information Center

Brabson, G. Dana; Waugh, David S.

1986-01-01

Describes procedures involved with paper electrophoresis separation of amino acids, gel electrophoresis separation of DNA, and design of an electrophoresis tank. Describes experiments using paper (amino acids) and gel (deoxyribonucleic acid fragments). Provides material lists, procedures, and discussion. (JM)

Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

USDA-ARS?s Scientific Manuscript database

The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

Rheological and structural characterization of agar/whey proteins insoluble complexes.

PubMed

Rocha, Cristina M R; Souza, Hiléia K S; Magalhães, Natália F; Andrade, Cristina T; Gonçalves, Maria Pilar

2014-09-22

Complex coacervation between whey proteins and carboxylated or highly sulphated polysaccharides has been widely studied. The aim of this work was to characterise a slightly sulphated polysaccharide (agar) and whey protein insoluble complexes in terms of yield, composition and physicochemical properties as well as to study their rheological behaviour for better understanding their structure. Unlike other sulphated polysaccharides, complexation of agar and whey protein at pH 3 in the absence of a buffering agent resulted in a coacervate that was a gel at 20°C with rheological properties and structure similar to those of simple agar gels, reinforced by proteins electrostatically aggregated to the agar network. The behaviour towards heat treatment was similar to that of agar alone, with a high thermal hysteresis and almost full reversibility. In the presence of citrate buffer, the result was a "flocculated solid", with low water content (75-81%), whose properties were governed by protein behaviour. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of the effect of power ultrasound on the nucleation of water during freezing of agar gel samples in tubing vials.

PubMed

Kiani, Hossein; Sun, Da-Wen; Delgado, Adriana; Zhang, Zhihang

2012-05-01

Nucleation, as an important stage of freezing process, can be induced by the irradiation of power ultrasound. In this study, the effect of irradiation temperature (-2 °C, -3 °C, -4 °C and -5 °C), irradiation duration (0s, 1s, 3s, 5s, 10s or 15s) and ultrasound intensity (0.07 W cm(-2), 0.14 W cm(-2), 0.25 W cm(-2), 0.35 W cm(-2) and 0.42 W cm(-2)) on the dynamic nucleation of ice in agar gel samples was studied. The samples were frozen in an ethylene glycol-water mixture (-20 °C) in an ultrasonic bath system after putting them into tubing vials. Results indicated that ultrasound irradiation is able to initiate nucleation at different supercooled temperatures (from -5 °C to -2 °C) in agar gel if optimum intensity and duration of ultrasound were chosen. Evaluation of the effect of 0.25 W cm(-2) ultrasound intensity and different durations of ultrasound application on agar gels showed that 1s was not long enough to induce nucleation, 3s induced the nucleation repeatedly but longer irradiation durations resulted in the generation of heat and therefore nucleation was postponed. Investigation of the effect of ultrasound intensity revealed that higher intensities of ultrasound were effective when a shorter period of irradiation was used, while lower intensities only resulted in nucleation when a longer irradiation time was applied. In addition to this, higher intensities were not effective at longer irradiation times due to the heat generated in the samples by the heating effect of ultrasound. In conclusion, the use of ultrasound as a means to control the crystallization process offers promising application in freezing of solid foods, however, optimum conditions should be selected. Copyright © 2011 Elsevier B.V. All rights reserved.

Simple and Rapid System for Two-Dimensional Gel Electrophoresis Technique: A Laboratory Exercise for High School Students

ERIC Educational Resources Information Center

Maurye, Praveen; Basu, Arpita; Biswas, Jayanta Kumar; Bandyopadhyay, Tapas Kumar; Naskar, Malay

2018-01-01

Polyacrylamide gel electrophoresis (PAGE) is the most classical technique favored worldwide for resolution of macromolecules in many biochemistry laboratories due to its incessant advanced developments and wide modifications. These ever-growing advancements in the basic laboratory equipments lead to emergence of many expensive, complex, and tricky…

Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis.

PubMed

Hayakawa, M; Hosogi, Y; Takiguchi, H; Saito, S; Shiroza, T; Shibata, Y; Hiratsuka, K; Kiyama-Kishikawa, M; Abiko, Y

2001-01-15

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step. Copyright 2001 Academic Press.

Getting the Most out of Electrophoresis Units

ERIC Educational Resources Information Center

Mulvihill, Charlotte

2007-01-01

At Oklahoma City Community College, they have developed gel electrophoresis activities that support active learning of many scientific concepts, including: pH, electrolysis, oxidation reduction, electrical currents, potentials, conductivity, molarity, gel electrophoresis, DNA and protein separation, and DNA fingerprinting. This article presents…

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

PubMed Central

Chen, J D; Denton, M J; Morgan, G; Pearn, J H; Mackinlay, A G

1988-01-01

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus. Images Figure 1 PMID:3358426

Theory of DNA electrophoresis in physical gels and entangled polymer solutions

NASA Astrophysics Data System (ADS)

Duke, Thomas; Viovy, Jean Louis

1994-03-01

A scaling theory is presented for the electrophoretic mobility of DNA in sieving media that form dynamically evolving meshworks, such as physical gels and solutions of entangled polymers. In such media, the topological constraints on the DNA's motion are perpetually changing as cross links break and rejoin or as the polymers diffuse. It is shown that if the rate of constraint release falls within a certain range (which depends on the field strength), fractionation can be extended to higher molecular weights than would be feasible using a permanent gel of equivalent pore size. This improvement is a consequence of the disruptive effect that constraint release has on the mechanism of molecular orientation. Numerical simulations support the predictions of the theory. The possibility of realizing such a system in practice, with the aim of improving on current electrophoresis methods, is commented upon. It is suggested that semidilute polymer solutions may be a versatile medium for the rapid separation of long single-stranded DNA molecules, and the particular quality of solution required is identified.

Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

ERIC Educational Resources Information Center

Kim, Thomas D.; Craig, Paul A.

2010-01-01

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

Recent advances in preparative electrophoresis

NASA Technical Reports Server (NTRS)

Mosher, Richard A.; Thormann, Wolfgang; Egen, Ned B.; Couasnon, Pascal; Sammons, David W.

1987-01-01

Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty

PubMed Central

Chiva, A

2013-01-01

Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty. Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants. Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins (acute phase proteins, albumin, lipoproteins, and immunoglobulins) by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results. Abbreviations CTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP

Small-scale enzymatic digestion of glycoproteins and proteoglycans for analysis of oligosaccharides by LC-MS and FACE gel electrophoresis.

PubMed

Estrella, Ruby P; Whitelock, John M; Roubin, Rebecca H; Packer, Nicolle H; Karlsson, Niclas G

2009-01-01

Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

Detection of mechanically recovered chicken meat using capillary gel electrophoresis.

PubMed

Day, L; Brown, H

2001-05-01

This study investigated the use of capillary gel electrophoresis (CGE) as a method for differentiating between raw mechanically recovered chicken meat (MRM) and hand deboned chicken breast meat (HDM). Twenty samples of MRM were obtained and twenty samples of HDM were prepared in the laboratory. They were extracted and analysed using Prosort™ SDS-protein analysis reagent. There were obvious differences in the relative peak areas within the profiles obtained which distinguished raw MRM from raw HDM; specifically, that of haemoglobin was higher in MRM. Using the peak area of haemoglobin and its ratio to other peaks, the technique was tested using composite MRM-HDM mixtures. The results suggest that it is possible to differentiate mixtures containing 7.5% MRM from that of 0% MRM using the CGE method.

Genetic diversity demonstrated by pulsed field gel electrophoresis of Salmonella enterica isolates obtained from diverse sources in Mexico

USDA-ARS?s Scientific Manuscript database

This study was conducted to determine the genetic diversity of Salmonella isolates recovered from a variety of sources using pulsed-field gel electrophoresis (PFGE) to assess their possible relatedness. Salmonella was isolated from ca. 52% of samples from a pepper var. Bell production system. A to...

WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins.

PubMed

Kubota, Yuji; Fujioka, Ko; Takekawa, Mutsuhiro

2017-01-01

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Identification of Methanococcus Jannaschii Proteins in 2-D Gel Electrophoresis Patterns by Mass Spectrometry

DOE R&D Accomplishments Database

Liang, X.

1998-06-10

The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

Apparatus for electrophoresis separation

DOEpatents

Anderson, Norman L.

1978-01-01

An apparatus is disclosed for simultaneously performing electrophoresis separations on a plurality of slab gels containing samples of protein, protein subunits or nucleic acids. A reservoir of buffer solution is divided into three compartments by two parallel partitions having vertical slots spaced along their length. A sheet of flexible, electrically insulative material is attached to each partition and is provided with vertical slits aligned with the slots. Slab-gel holders are received within the slots with the flexible material folded outwardly as flaps from the slits to overlay portions of the holder surfaces and thereby act as electrical and liquid seals. An elongated, spaghetti-like gel containing a sample of specimen that was previously separated by isoelectric focusing techniques is vertically positioned along a marginal edge portion of the slab gel. On application of an electrical potential between the two outer chambers of buffer solution, a second dimensional electrophoresis separation in accordance with molecular weight occurs as the specimen molecules migrate across the slab gel.

Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

PubMed Central

Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

1974-01-01

By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

Affinity in electrophoresis.

PubMed

Heegaard, Niels H H

2009-06-01

The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

Accommodating brightness and exposure levels in densitometry of stained polyacrylamide electrophoresis gels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Han Yen; Ng, Tuck Wah; Liew, Oi Wah

2010-03-20

Flatbed scanner densitometers can be operated under various illumination and recording exposure levels. In this work, we show that optical density measurement accuracy, sensitivity, and stability of stained polyacrylamide electrophoresis gel densitometry are crucially dependent on these two factors (brightness and exposure level), notwithstanding that the source is monochromatic, spatially uniform, and the measurements are made using an accurately calibrated step wedge in tandem. We further outline a method to accommodate the intensity deviations over a range of illumination and exposure levels in order to maintain sensitivity and repeatability in the computed optical densities. Comparisons were also made with resultsmore » from a commercial densitometer.« less

The use of the 2-aminobenzoic acid tag for oligosaccharide gel electrophoresis.

PubMed

Huang, Z; Prickett, T; Potts, M; Helm, R F

2000-08-18

Gel electrophoresis of fluorophore labeled saccharides provides a rapid and reliable method to screen enzymatic and/or chemical treatments of polysaccharides and glycoconjugates, as well as a sensitive and efficient microscale method to separate and purify oligosaccharides for further analysis. A simple and inexpensive method of derivatization and analysis using 2-aminobenzoic acid (anthranilic acid, AA) is described and applied to the extracellular polysaccharide released by the desiccation tolerant cyanobacterium Nostoc commune DRH-1. The results of these analyses suggest a possible protective functionality of two pendent groups, as well as a potential relationship between these groups and the desiccation tolerance of the organism.

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.

Photothermal characterization of the gelation process in Gelidium robustum Agar

NASA Astrophysics Data System (ADS)

Freile-Pelegrín, Y.; Bante, J.; Alvarado-Gil, J. J.; Yánez-Limón, J. M.

2005-06-01

Agar is a hydrophilic colloid formed by polysaccharides, whose ability to form reversible gels simply by cooling hot aqueous solutions is the most important property and can be regarded as the prototype and model for all gelling systems. In this paper the evolution of the gelation process of agar obtained from algae of the species Gelidium robustum, using the photopyroelectric technique is reported. It is shown that thermal effusivity increase when the agar is cooled, reaching a maximum value around 37°C. The increase in thermal effusivity can be related to the increasing of the bondings in the gel as temperature decreases, reaching the maximum at the gelation point. The decrease of the thermal effusivity at lower temperature could be due to the syneresis process involving a gradual release of water after gelation.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue.

PubMed

Pennington, Kyla; McGregor, Emma; Beasley, Clare L; Everall, Ian; Cotter, David; Dunn, Michael J

2004-01-01

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Migration of Chemotactic Bacteria in Soft Agar: Role of Gel Concentration

PubMed Central

Croze, Ottavio A.; Ferguson, Gail P.; Cates, Michael E.; Poon, Wilson C.K.

2011-01-01

We study the migration of chemotactic wild-type Escherichia coli populations in semisolid (soft) agar in the concentration range C = 0.15–0.5% (w/v). For C≲0.35%, expanding bacterial colonies display characteristic chemotactic rings. At C = 0.35%, however, bacteria migrate as broad circular bands rather than sharp rings. These are growth/diffusion waves arising because of suppression of chemotaxis by the agar and have not been previously reported experimentally to our knowledge. For C = 0.4–0.5%, expanding colonies do not span the depth of the agar and develop pronounced front instabilities. The migration front speed is weakly dependent on agar concentration at C < 0.25%, but decreases sharply above this value. We discuss these observations in terms of an extended Keller-Segel model for which we derived novel transport parameter expressions accounting for perturbations of the chemotactic response by collisions with the agar. The model makes it possible to fit the observed front speed decay in the range C = 0.15–0.35%, and its solutions qualitatively reproduce the observed transition from chemotactic to growth/diffusion bands. We discuss the implications of our results for the study of bacteria in porous media and for the design of improved bacteriological chemotaxis assays. PMID:21806920

A comparison of non-typhoidal Salmonella from humans and food animals using pulsed-field gel electrophoresis and antimicrobial susceptibility patterns

USDA-ARS?s Scientific Manuscript database

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoida...

Combination of Multiplex PCR and PCR-Denaturing Gradient Gel Electrophoresis for Monitoring Common Sourdough-Associated Lactobacillus Species

PubMed Central

Settanni, Luca; Valmorri, Sara; van Sinderen, Douwe; Suzzi, Giovanna; Paparella, Antonello; Corsetti, Aldo

2006-01-01

A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs. PMID:16672538

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.

Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations.

PubMed

Day, I N; O'Dell, S D; Spanakis, E; Weavind, G P

1999-02-01

Important requirements for molecular genetic epidemiological studies are economy, sample parallelism, convenience of setup and accessibility, goals inadequately met by existent approaches. We invented microplate array diagonal gel electrophoresis (MADGE) to gain simultaneously the advantages of simple setup, 96-well microplate compatibility, horizontal electrophoresis, and the resolution of polyacrylamide. At essentially no equipment cost (one simple plastic gel former), 10-100-fold savings on time for sample coding, liquid transfers, and data documentation, in addition to volume reductions and gel re-use, can be achieved. MADGE is compatible with ARMS, restriction analysis and other pattern analyses. CpG-PCR is a general PCR approach to CpG sites (10-20% of all human single base variation): both primers have 3' T, and are abutted to the CpG, forcing a TaqI restriction site if the CpG is intact. Typically, a 52 bp PCR product is then cut in half. CpG-PCR also illustrates that PAGE-MADGE readily permits analysis of 'ultrashort' PCRs. Melt-MADGE employs real-time-variable-temperature electrophoresis to examine duplex mobility during melting, achieving DGGE-like de novo, mutation scanning, but with the conveniences of arbitrary programmability, MADGE compatibility and short run time. This suite of methods enhances our capability to type or scan thousands of samples simultaneously, by 10-100-fold.

Influence of the extraction process on the rheological and structural properties of agars.

PubMed

Sousa, Ana M M; Borges, João; Silva, A Fernando; Gonçalves, Maria P

2013-07-01

Agars obtained by traditional hot-water (TWE) and microwave-assisted (MAE) extractions were compared in terms of their rheological and physicochemical properties and molecular self-association in solutions of low (0.05%, w/w) and high (1.5%, w/w) polymer concentrations. At low concentration, thin gelled layers were imaged by AFM. Slow or rapid cooling of the solutions influenced structure formation. In each case, TWE and MAE agar structures were different and apparently larger for MAE. At high concentration, progressive structural reinforcement was seen; while TWE agar showed a more open and irregular 3D network, MAE agar gel imaged by cryoSEM was denser and fairly uniform. The rheological (higher thermal stability and consistency) and mechanical (higher gel strength) behaviors of MAE agar seemed consistent with a positive effect of molecular mass and 3,6-anhydro-α-l-galactose content. MAE produced non-degraded agar comparable with commercial ones and if properly monitored, could be a promising alternative to TWE. Copyright © 2013 Elsevier Ltd. All rights reserved.

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

PubMed Central

Walter, Jens; Hertel, Christian; Tannock, Gerald W.; Lis, Claudia M.; Munro, Karen; Hammes, Walter P.

2001-01-01

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects. PMID:11375166

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

PubMed

Raghupathy, V; Oommen, Anna; Ramachandran, Anup

2014-06-15

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

Separation of oligopeptides, nucleobases, nucleosides and nucleotides using capillary electrophoresis/electrochromatography with sol-gel modified inner capillary wall.

PubMed

Svobodová, Jana; Kofroňová, Olga; Benada, Oldřich; Král, Vladimír; Mikšík, Ivan

2017-09-29

The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides. Copyright © 2017 Elsevier B.V. All rights reserved.

Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus.

PubMed

Laarhoven, Bob; Elissen, H J H; Temmink, H; Buisman, C J N

2016-01-01

An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml), 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin). With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates.

Agar Sediment Test for Assessing the Suitability of Organic Waste Streams for Recovering Nutrients by the Aquatic Worm Lumbriculus variegatus

PubMed Central

Laarhoven, Bob; Elissen, H. J. H.; Temmink, H.; Buisman, C. J. N.

2016-01-01

An agar sediment test was developed to evaluate the suitability of organic waste streams from the food industry for recovering nutrients by the aquatic worm Lumbriculus variegatus (Lv). The effects of agar gel, sand, and food quantities in the sediment test on worm growth, reproduction, and water quality were studied. Agar gel addition ameliorated growth conditions by reducing food hydrolysis and altering sediment structure. Best results for combined reproduction and growth were obtained with 0.6% agar-gel (20 ml), 10 g. fine sand, 40 g. coarse sand, and 105 mg fish food (Tetramin). With agar gel, ingestion and growth is more the result of addition of food in its original quality. Final tests with secondary potato starch sludge and wheat bran demonstrated that this test is appropriate for the comparison of solid feedstuffs and suspended organic waste streams. This test method is expected to be suitable for organic waste studies using other sediment dwelling invertebrates. PMID:26937632

Proton beam writing of microstructures in Agar gel for patterned cell growth

NASA Astrophysics Data System (ADS)

Larisch, Wolfgang; Koal, Torsten; Werner, Ronald; Hohlweg, Marcus; Reinert, Tilo; Butz, Tilman

2011-10-01

A rather useful prerequisite for many biological and biophysical studies, e.g., for cell-cell communication or neuronal networks, is confined cell growth on micro-structured surfaces. Solidified Agar layers have smooth surfaces which are electrically neutral and thus inhibit receptor binding and cell adhesion. For the first time, Agar microstructures have been manufactured using proton beam writing (PBW). In the irradiated Agar material the polysaccharides are split into oligosaccharides which can easily be washed off leaving Agar-free areas for cell adhesion. The beam diameter of 1 μm allows the fabrication of compartments accommodating single cells which are connected by micrometer-sized channels. Using the external beam the production process is very fast. Up to 50 Petri dishes can be produced per day which makes this technique very suitable for biological investigations which require large throughputs.

Optimizing photovoltaic performance in CuInS 2 and CdS quantum dot-sensitized solar cells by using an agar-based gel polymer electrolyte

DOE PAGES

Raphael, E.; Jara, D. H.; Schiavon, M. A.

2017-01-19

Quantum dot-sensitized solar cells (QDSSCs) offer new opportunities to address the clean energy challenge, being one of the top candidates for third generation photovoltaics. Like dye-sensitized solar cells (DSSCs), QDSSCs normally use liquid electrolytes that suffer from issues such as evaporation or leakage. In this study a gel polysulfide electrolyte was prepared containing a natural polymer, agar, and was used as a quasi-solid-state electrolyte in solar cells to replace the conventional liquid electrolytes. This gel electrolyte shows almost the same conductivity as the liquid one. The solar cells were fabricated using CuInS 2 quantum dots (QDs), previously synthesized, deposited onmore » TiO 2 photoanodes by electrophoretic deposition (EPD). CdS was deposited on TiO 2 by successive ionic layer adsorption and reaction (SILAR). Reduced graphene oxide (RGO)–Cu 2S, brass, and thin film CuxS were used as counter electrodes. Compared to a liquid polysulfide water based electrolyte, solar cells based on CuInS 2 and CdS using gel polymer electrolyte (GPE) exhibit greater incident photon to current conversion efficiency (IPCE = 51.7% at 520 nm and 72.7% at 440 nm), photocurrent density (J sc = 10.75 and 13.51 mA cm -2), and power conversion efficiency (η = 2.97 and 2.98%) while exhibiting significantly enhanced stability. The solar cells employing the agar-based gel polymeric electrolyte are about a factor of 0.20 more stable than using a liquid electrolyte. The higher photovoltaic performance is due to the good conductivity and high wettability as well as the superior permeation capability of the gel electrolyte into the mesoporous matrix of a TiO 2 film« less

Optimizing photovoltaic performance in CuInS 2 and CdS quantum dot-sensitized solar cells by using an agar-based gel polymer electrolyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raphael, E.; Jara, D. H.; Schiavon, M. A.

Quantum dot-sensitized solar cells (QDSSCs) offer new opportunities to address the clean energy challenge, being one of the top candidates for third generation photovoltaics. Like dye-sensitized solar cells (DSSCs), QDSSCs normally use liquid electrolytes that suffer from issues such as evaporation or leakage. In this study a gel polysulfide electrolyte was prepared containing a natural polymer, agar, and was used as a quasi-solid-state electrolyte in solar cells to replace the conventional liquid electrolytes. This gel electrolyte shows almost the same conductivity as the liquid one. The solar cells were fabricated using CuInS 2 quantum dots (QDs), previously synthesized, deposited onmore » TiO 2 photoanodes by electrophoretic deposition (EPD). CdS was deposited on TiO 2 by successive ionic layer adsorption and reaction (SILAR). Reduced graphene oxide (RGO)–Cu 2S, brass, and thin film CuxS were used as counter electrodes. Compared to a liquid polysulfide water based electrolyte, solar cells based on CuInS 2 and CdS using gel polymer electrolyte (GPE) exhibit greater incident photon to current conversion efficiency (IPCE = 51.7% at 520 nm and 72.7% at 440 nm), photocurrent density (J sc = 10.75 and 13.51 mA cm -2), and power conversion efficiency (η = 2.97 and 2.98%) while exhibiting significantly enhanced stability. The solar cells employing the agar-based gel polymeric electrolyte are about a factor of 0.20 more stable than using a liquid electrolyte. The higher photovoltaic performance is due to the good conductivity and high wettability as well as the superior permeation capability of the gel electrolyte into the mesoporous matrix of a TiO 2 film« less

Quantitative gel electrophoresis: new records in precision by elaborated staining and detection protocols.

PubMed

Deng, Xi; Schröder, Simone; Redweik, Sabine; Wätzig, Hermann

2011-06-01

Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

PubMed Central

Tamura, A; Ohashi, N; Urakami, H; Takahashi, K; Oyanagi, M

1985-01-01

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera. Images PMID:3922893

Improving agar electrospinnability with choline-based deep eutectic solvents.

PubMed

Sousa, Ana M M; Souza, Hiléia K S; Uknalis, Joseph; Liu, Shih-Chuan; Gonçalves, Maria P; Liu, LinShu

2015-09-01

Very recently our group has produced novel agar-based fibers by an electrospinning technique using water as solvent and polyvinyl alcohol (PVA) as co-blending polymer. Here, we tested the deep eutectic solvent (DES), (2-hydroxyethyl)trimethylammonium chloride/urea prepared at 1:2 molar ratio, as an alternative solvent medium for agar electrospinning. The electrospun materials were collected with an ethanol bath adapted to a previous electrospinning set-up. One weight percent agar-in-DES showed improved viscoelasticity and hence, spinnability, when compared to 1 wt% agar-in-water and pure agar nanofibers were successfully electrospun if working above the temperature of sol-gel transition (∼80 °C). By changing the solvent medium we decreased the PVA concentration (5 wt% starting solution) and successfully produced composite fibers with high agar contents (50/50 agar/PVA). Best composite fibers were formed with the 50/50 and 30/70 agar/PVA solutions. These fibers were mechanically resistant, showed tailorable surface roughness and diverse size distributions, with most of the diameters falling in the sub-micron range. Both nano and micro forms of agar fibers (used separately or combined) may have potential for the design of new and highly functional agar-based materials. Published by Elsevier B.V.

The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

NASA Technical Reports Server (NTRS)

Zieboll, Gerhard; Orgel, Leslie E.

1994-01-01

We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

Sample collection system for gel electrophoresis

DOEpatents

Olivares, Jose A.; Stark, Peter C.; Dunbar, John M.; Hill, Karen K.; Kuske, Cheryl R.; Roybal, Gustavo

2004-09-21

An automatic sample collection system for use with an electrophoretic slab gel system is presented. The collection system can be used with a slab gel have one or more lanes. A detector is used to detect particle bands on the slab gel within a detection zone. Such detectors may use a laser to excite fluorescently labeled particles. The fluorescent light emitted from the excited particles is transmitted to low-level light detection electronics. Upon the detection of a particle of interest within the detection zone, a syringe pump is activated, sending a stream of buffer solution across the lane of the slab gel. The buffer solution collects the sample of interest and carries it through a collection port into a sample collection vial.

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

PubMed

Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

2005-09-01

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing.

PubMed

Lindstedt, Bjørn-Arne; Tham, Wilhelm; Danielsson-Tham, Marie-Louise; Vardund, Traute; Helmersson, Seved; Kapperud, Georg

2008-02-01

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Simulating Electrophoresis.

ERIC Educational Resources Information Center

Moertel, Cheryl; Frutiger, Bruce

1996-01-01

Describes a DNA fingerprinting simulation that uses vegetable food coloring and plastic food containers instead of DNA and expensive gel electrophoresis chambers. Allows students to decipher unknown combinations of dyes in a method similar to that used to decipher samples of DNA in DNA fingerprint techniques. (JRH)

Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

PubMed

Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

2016-02-11

Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

PubMed Central

Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

2016-01-01

Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

2016-02-01

Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

The laboratory technology of discrete molecular separation: the historical development of gel electrophoresis and the material epistemology of biomolecular science, 1945-1970.

PubMed

Chiang, Howard Hsueh-hao

2009-01-01

Preparative and analytical methods developed by separation scientists have played an important role in the history of molecular biology. One such early method is gel electrophoresis, a technique that uses various types of gel as its supporting medium to separate charged molecules based on size and other properties. Historians of science, however, have only recently begun to pay closer attention to this material epistemological dimension of biomolecular science. This paper substantiates the historiographical thread that explores the relationship between modern laboratory practice and the production of scientific knowledge. It traces the historical development of gel electrophoresis from the mid-1940s to the mid-1960s, with careful attention to the interplay between technical developments and disciplinary shifts, especially the rise of molecular biology in this time-frame. Claiming that the early 1950s marked a decisive shift in the evolution of electrophoretic methods from moving boundary to zone electrophoresis, I reconstruct various trajectories in which scientists such as Oliver Smithies sought out the most desirable solid supporting medium for electrophoretic instrumentation. Biomolecular knowledge, I argue, emerged in part from this process of seeking the most appropriate supporting medium that allowed for discrete molecular separation and visualization. The early 1950s, therefore, marked not only an important turning point in the history of separation science, but also a transformative moment in the history of the life sciences as the growth of molecular biology depended in part on the epistemological access to the molecular realm available through these evolving technologies.

Electrophoresis Gel Quantification with a Flatbed Scanner and Versatile Lighting from a Screen Scavenged from a Liquid Crystal Display (LCD) Monitor

ERIC Educational Resources Information Center

Yeung, Brendan; Ng, Tuck Wah; Tan, Han Yen; Liew, Oi Wah

2012-01-01

The use of different types of stains in the quantification of proteins separated on gels using electrophoresis offers the capability of deriving good outcomes in terms of linear dynamic range, sensitivity, and compatibility with specific proteins. An inexpensive, simple, and versatile lighting system based on liquid crystal display backlighting is…

Agar-Silica-Gel Heating Phantom May Be Suitable for Long-Term Quality Assurance of MRgHIFU

NASA Astrophysics Data System (ADS)

Partanen, Ari

2009-04-01

In MRgHIFU, the purpose of frequent quality assurance is to detect changes in system performance to prevent adverse effects during treatments. Due to high ultrasound intensities in MRgHIFU, it is essential to assure that the procedure is safe and efficacious and that image-based guidance of the treatment is reliable. We aimed to develop a guideline for MRgHIFU QA by acquiring MR temperature maps during ultrasonic heating of an agar-silica-gel phantom over a four month-period using three separate MRgHIFU uterine leiomyoma treatment systems. From this data, the stability of the maximum temperature elevation, the targeting accuracy, and the dimensions of the heated volume were analyzed. Additionally, we studied the sensitivity of these parameters to reveal hypothetical decrease in HIFU performance. After calibration, the mean targeting offsets of the heated volume were observed to be less than 2 mm in the three orthogonal directions. The measured maximum temperature elevation and the length and the width of the heated volume remained consistent throughout the four-month period. Furthermore, it was found that the parameters under investigation were sensitive to reveal the decreased HIFU performance. We conclude that an agar-silica -based phantom is suitable for targeting accuracy and heating properties QA of MRgHIFU system even in long-term use. Moreover, this simple QA method may be used to reveal small changes in HIFU performance assuring consistent functionality and safety of the MRgHIFU system.

Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

PubMed Central

Temmerman, R.; Scheirlinck, I.; Huys, G.; Swings, J.

2003-01-01

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential. PMID:12513998

Synthesis of hydrogel via click chemistry for DNA electrophoresis.

PubMed

Finetti, Chiara; Sola, Laura; Elliott, Jim; Chiari, Marcella

2017-09-01

This work introduces a novel sieving gel for DNA electrophoresis using a classical click chemistry reaction, the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), to cross-link functional polymer chains. The efficiency of this reaction provides, under mild conditions, hydrogels with near-ideal network connectivity and improved physical properties. Hydrogel formation via click chemistry condensation of functional polymers does not involve the use of toxic monomers and UV initiation. The performance of the new hydrogel in the separation of double stranded DNA fragments was evaluated in the 2200 TapeStation system, an analytical platform, recently introduced by Agilent that combines the advantages of CE in terms of miniaturization and automation with the simplicity of use of slab gel electrophoresis. The click gel enables addition of florescent dyes prior to electrophoresis with considerable improvement of resolution and separation efficiency over conventional cross-linked polyacrylamide gels. Copyright © 2017 Elsevier B.V. All rights reserved.

Case study of the use of pulsed field gel electrophoresis in the detection of a food-borne outbreak.

PubMed

De Lappe, Niall; Cormican, Martin

2015-01-01

In early July 2008, a cluster of six Salmonella Agona was identified in the Republic of Ireland. A dispersed, common source outbreak was suspected. Later in July a further case was identified and the Health Protection Agency in the UK indicated that they had 32 cases of S. Agona since Feb 2008. This chapter discusses how pulsed field gel electrophoresis was used to help confirm an outbreak and to trace the source of the outbreak.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

PubMed

Krizek, D R; Rick, M E

2000-03-15

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

PubMed Central

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.

2011-01-01

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were 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, and 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. PMID:21684248

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.

PubMed

Quinn, Robert A; Stevenson, Roselynn M W

2012-05-01

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.

Two-Dimensional Gel Electrophoresis: Discovering Biomolecules for Environmental Bioremediation

NASA Astrophysics Data System (ADS)

Singh, Om V.; Chandel, Anuj K.

Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a “perfect medicine.” The solution to the problems with bioremediation lies in analyzing to what extent the microbes’ physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensional gel electrophoresis (2DE) in conjunction with mass spectrometry (MS) can be utilized to identify the biomolecules and their molecular mechanisms in bioremediation. A set of highly abundant global proteins over a pI range 4-7 was separated and compared by size fractionation (25-100 kDa) on 2DE. We identified a set of catabolic proteins, enzymes, and heat shock molecular chaperones associated with the regulatory network that was found to be overexpressed under phenol-stressed conditions. This chapter also offers optimized ideal directions for 2DE, followed by easy-to-follow directions for a protein identification strategy using MALDI-TOF and targeting novel proteins/enzymes for a universal set of experiments.

21 CFR 582.7115 - Agar-agar.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Agar-agar. 582.7115 Section 582.7115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7115 Agar-agar. (a...

21 CFR 582.7115 - Agar-agar.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Agar-agar. 582.7115 Section 582.7115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Stabilizers § 582.7115 Agar-agar. (a...

Visualization of DNA molecules in time during electrophoresis

NASA Technical Reports Server (NTRS)

Lubega, Seth

1991-01-01

For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

Development of an open source laboratory information management system for 2-D gel electrophoresis-based proteomics workflow

PubMed Central

Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa

2006-01-01

Background In the post-genome era, most research scientists working in the field of proteomics are confronted with difficulties in management of large volumes of data, which they are required to keep in formats suitable for subsequent data mining. Therefore, a well-developed open source laboratory information management system (LIMS) should be available for their proteomics research studies. Results We developed an open source LIMS appropriately customized for 2-D gel electrophoresis-based proteomics workflow. The main features of its design are compactness, flexibility and connectivity to public databases. It supports the handling of data imported from mass spectrometry software and 2-D gel image analysis software. The LIMS is equipped with the same input interface for 2-D gel information as a clickable map on public 2DPAGE databases. The LIMS allows researchers to follow their own experimental procedures by reviewing the illustrations of 2-D gel maps and well layouts on the digestion plates and MS sample plates. Conclusion Our new open source LIMS is now available as a basic model for proteome informatics, and is accessible for further improvement. We hope that many research scientists working in the field of proteomics will evaluate our LIMS and suggest ways in which it can be improved. PMID:17018156

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

NASA Technical Reports Server (NTRS)

Sutherland, Betsy M.; Georgakilas, Alexandros G.; Bennett, Paula V.; Laval, Jacques; Sutherland, John C.; Gewirtz, A. M. (Principal Investigator)

2003-01-01

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

Comparative two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of human milk to identify dysregulated proteins in breast cancer.

PubMed

Aslebagh, Roshanak; Channaveerappa, Devika; Arcaro, Kathleen F; Darie, Costel C

2018-05-13

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC-MS/MS) analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses.

PubMed

Shiraishi, Rikiya; Nishiguchi, Akiko; Tsukamoto, Kenji; Muramatsu, Masatake

2012-09-01

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

Genotoxic potential of bee venom (Apis Mellifera) on human peripheral blood lymphocytes in vitro using single cell gel electrophoresis assay.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2008-09-01

Bee venom (BV) has been known to have therapeutic applications in traditional medicine to treat variety of diseases. It is also known that bee venom possesses anti-inflammatory and anticancer effects and that it can inhibit proliferation and induces apoptosis in cancer cells, but there is lack of information regarding genotoxicity of whole bee venom on normal human cells. In the present study, peripheral blood human lymphocytes from healthy donor were exposed in vitro to different concentration (5, 10, 25, 50 and 100 micro g/mL) of whole bee venom at different time periods (1, 6 and 24 hours). The single cell gel electrophoresis (SCGE) assay was used to evaluate the genotoxicity towards human cells. Results showed statistically significant increase in DNA damage caused in BV treated human lymphocytes compared to corresponding control cells for the tail length and tail moment. These results show that the extent of DNA damage, determined by the use of single cell gel electrophoresis is time and dose dependent. Based on the results it is clear that whole bee venom induces DNA damage and has genotoxic potential on human peripheral blood lymphocytes in vitro.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

PubMed Central

Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

2016-01-01

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

PubMed

dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

2012-01-27

Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

Aeromonas isolates from human diarrheic stool and groundwater compared by pulsed-field gel electrophoresis.

PubMed

Borchardt, Mark A; Stemper, Mary E; Standridge, Jon H

2003-02-01

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients' drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.

Automatic multiple-sample applicator and electrophoresis apparatus

NASA Technical Reports Server (NTRS)

Grunbaum, B. W. (Inventor)

1977-01-01

An apparatus for performing electrophoresis and a multiple-sample applicator is described. Electrophoresis is a physical process in which electrically charged molecules and colloidal particles, upon the application of a dc current, migrate along a gel or a membrane that is wetted with an electrolyte. A multiple-sample applicator is provided which coacts with a novel tank cover to permit an operator either to depress a single button, thus causing multiple samples to be deposited on the gel or on the membrane simultaneously, or to depress one or more sample applicators separately by means of a separate button for each applicator.

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

PubMed

Zhu, X Q; Chilton, N B; Gasser, R B

1998-05-01

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.

Estimating the Diffusion Coefficients of Sugars Using Diffusion Experiments in Agar-Gel and Computer Simulations.

PubMed

Miyamoto, Shuichi; Atsuyama, Kenji; Ekino, Keisuke; Shin, Takashi

2018-01-01

The isolation of useful microbes is one of the traditional approaches for the lead generation in drug discovery. As an effective technique for microbe isolation, we recently developed a multidimensional diffusion-based gradient culture system of microbes. In order to enhance the utility of the system, it is favorable to have diffusion coefficients of nutrients such as sugars in the culture medium beforehand. We have, therefore, built a simple and convenient experimental system that uses agar-gel to observe diffusion. Next, we performed computer simulations-based on random-walk concepts-of the experimental diffusion system and derived correlation formulas that relate observable diffusion data to diffusion coefficients. Finally, we applied these correlation formulas to our experimentally-determined diffusion data to estimate the diffusion coefficients of sugars. Our values for these coefficients agree reasonably well with values published in the literature. The effectiveness of our simple technique, which has elucidated the diffusion coefficients of some molecules which are rarely reported (e.g., galactose, trehalose, and glycerol) is demonstrated by the strong correspondence between the literature values and those obtained in our experiments.

Gel electrophoresis of partially denatured DNA. Retardation effect: its analysis and application.

PubMed Central

Lyamichev, V I; Panyutin, I G; Lyubchenko YuL

1982-01-01

The hypothesis about the role of partial denaturation in DNA retardation during its electrophoresis in denaturing gel /1,2/ was tested. We used partially melted DNA molecules in which the size of the melted regions and their location were known. They were obtained through glyoxal treatment of the melted regions by a procedure allowing the denatured state to be fixed at any point within the melting range. The approach and the availability of the melting maps of DNAs made it possible to investigate DNA molecules differing in length and in the size of the melted regions. The presence of a denatured region at the end of the molecule or inside of it was shown to decrease its electrophoretic mobility, the effect depending on the size of the melted region and on the DNA length. On the basis of the experimental results an explanation is proposed for the cause of retardation in the case of partially denatured DNA. Images PMID:7133999

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins

PubMed Central

Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

2017-01-01

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

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.

Counterion dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic gel digestion of stained protein for mass spectrometry.

PubMed

Cong, Wei-Tao; Wang, Xu; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

2012-01-01

A fast and matrix-assisted laser desorption/ionization mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet, to form an ion-pair complex. The protocol, including fixing, staining, and quick washing steps, can be completed in 1-1.5 h, depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8 ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

PubMed

Papalexandratou, Zoi; De Vuyst, Luc

2011-11-01

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Coagulase-negative staphylococci from non-mastitic bovine mammary gland: characterization of Staphylococcus chromogenes and Staphylococcus haemolyticus by antibiotic susceptibility testing and pulsed-field gel electrophoresis.

PubMed

Pate, Mateja; Zdovc, Irena; Avberšek, Jana; Ocepek, Matjaž; Pengov, Andrej; Podpečan, Ožbalt

2012-05-01

During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (28·8%), followed by Staph. simulans (11·2%), Staph. xylosus (11·2%), Staph. epidermidis (7·5%), Staph. hyicus (6·3%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation.

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

PubMed

Quero, Sara; García-Núñez, Marian; Párraga-Niño, Noemí; Barrabeig, Irene; Pedro-Botet, Maria L; de Simon, Mercè; Sopena, Nieves; Sabrià, Miquel

2016-06-01

To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

Aeromonas Isolates from Human Diarrheic Stool and Groundwater Compared by Pulsed-Field Gel Electrophoresis

PubMed Central

Stemper, Mary E.; Standridge, Jon H.

2003-01-01

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients’ drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas. Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas-positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures. PMID:12603994

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

PubMed

Kotásková, Iva; Mališová, Barbora; Obručová, Hana; Holá, Veronika; Peroutková, Tereza; Růžička, Filip; Freiberger, Tomáš

2017-01-01

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment. © 2017 S. Karger AG, Basel.

Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

PubMed

Ercolini, D; Moschetti, G; Blaiotta, G; Coppola, S

2001-03-01

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data.

PubMed

Eddhif, Balkis; Guignard, Nadia; Batonneau, Yann; Clarhaut, Jonathan; Papot, Sébastien; Geffroy-Rodier, Claude; Poinot, Pauline

2018-04-01

The data presented here are related to the research paper entitled "Study of a Novel Agent for TCA Precipitated Proteins Washing - Comprehensive Insights into the Role of Ethanol/HCl on Molten Globule State by Multi-Spectroscopic Analyses" (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

Tunable separations based on a molecular size effect for biomolecules by poly(ethylene glycol) gel-based capillary electrophoresis.

PubMed

Kubo, Takuya; Nishimura, Naoki; Furuta, Hayato; Kubota, Kei; Naito, Toyohiro; Otsuka, Koji

2017-11-10

We report novel capillary gel electrophoresis (CGE) with poly(ethylene glycol) (PEG)-based hydrogels for the effective separations of biomolecules containing sugars and DNAs based on a molecular size effect. The gel capillaries were prepared in a fused silica capillary modified with 3-(trimethoxysilyl)propylmethacrylate using a variety of the PEG-based hydrogels. After the fundamental evaluations in CGE regarding the separation based on the molecular size effect depending on the crosslinking density, the optimized capillary provided the efficient separation of glucose ladder (G1 to G20). In addition, another capillary showed the successful separation of DNA ladder in the range of 10-1100 base pair, which is superior to an authentic acrylamide-based gel capillary. For both glucose and DNA ladders, the separation ranges against the molecular size were simply controllable by alteration of the concentration and/or units of ethylene oxide in the PEG-based crosslinker. Finally, we demonstrated the separations of real samples, which included sugars carved out from monoclonal antibodies, mAbs, and then the efficient separations based on the molecular size effect were achieved. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.

PubMed

Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; de Gaetano, Giovanni; Rotilio, Domenico

2008-02-01

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.

Analysis of bacterial populations in the environment using two-dimensional gel electrophoresis of genomic DNA and complementary DNA.

PubMed

Liu, Guo-Hua; Nakamura, Tatsuo; Amemiya, Takashi; Rajendran, Narasimmalu; Itoh, Kiminori

2011-01-01

Two-dimensional gel electrophoresis (2-DGE) mapping of genomic DNA and complementary DNA (cDNA) amplicons was attempted to analyze total and active bacterial populations within soil and activated sludge samples. Distinct differences in the number and species of bacterial populations and those that were metabolically active at the time of sampling were visually observed especially for the soil community. Statistical analyses and sequencing based on the 2-DGE data further revealed the relationships between total and active bacterial populations within each community. This high-resolution technique would be useful for obtaining a better understanding of bacterial population structures in the environment.

Urine Collected From Diapers Can Be Used for 2-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) in Infants and Young Children

PubMed Central

Kennedy, Mary Jayne; Griffin, Angela; Su, Ruifeng; Merchant, Michael; Klein, Jon

2011-01-01

Urinary proteomic profiling has potential to identify candidate biomarkers of renal injury in infants provided an adequate urine sample can be obtained. Although diapers are used to obtain urine for clinical evaluation, their use for proteomic analysis has not been investigated. We therefore performed feasibility studies on the use of diaper-extracted urine for 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Pediatric waste urine (2–20 mL) was applied to gel-containing, non-gel and cotton-gauze diapers and then mechanically expressed. Urine volume and total protein were measured pre- and post-extraction. Proteins were separated via 2D-PAGE following application of urine (20–40 mL) to each matrix. 2D-PAGE was also performed on clinical specimens collected using each diaper type. Differences in the adsorption and retention of urine volume and protein were noted between matrices. Non-gel and cotton-gauze diapers provided the best protein/volume recovery and the lowest interference with the Bradford assay. 2D-PAGE was also successfully completed using urine samples from both cotton fiber matrices. Conversely, samples from low-gel diapers demonstrated poor protein separation and reproducibility. Diapers containing cotton-fiber matrices appear adequate for 2D-PAGE. Qualitative and quantitative analyses of resolved proteins using replicate, high resolution gels will be required, however, before diaper-extracted urine can be applied in proteomic profiling. PMID:21137001

Identification of Fur, Aconitase, and Other Proteins Expressed by Mycobacterium tuberculosis under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry

PubMed Central

Wong, Diane K.; Lee, Bai-Yu; Horwitz, Marcus A.; Gibson, Bradford W.

1999-01-01

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance. PMID:9864233

Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M.; Zhan, Xianquan

2015-12-01

Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

PubMed

Xia, Minghui; Qi, Qingguo

2013-01-01

We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P < 0.01), there was no positive correlation; similarity (Dice coefficient) was 13.1% to 62.5%. Some bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

Spot quantification in two dimensional gel electrophoresis image analysis: comparison of different approaches and presentation of a novel compound fitting algorithm

PubMed Central

2014-01-01

Background Various computer-based methods exist for the detection and quantification of protein spots in two dimensional gel electrophoresis images. Area-based methods are commonly used for spot quantification: an area is assigned to each spot and the sum of the pixel intensities in that area, the so-called volume, is used a measure for spot signal. Other methods use the optical density, i.e. the intensity of the most intense pixel of a spot, or calculate the volume from the parameters of a fitted function. Results In this study we compare the performance of different spot quantification methods using synthetic and real data. We propose a ready-to-use algorithm for spot detection and quantification that uses fitting of two dimensional Gaussian function curves for the extraction of data from two dimensional gel electrophoresis (2-DE) images. The algorithm implements fitting using logical compounds and is computationally efficient. The applicability of the compound fitting algorithm was evaluated for various simulated data and compared with other quantification approaches. We provide evidence that even if an incorrect bell-shaped function is used, the fitting method is superior to other approaches, especially when spots overlap. Finally, we validated the method with experimental data of urea-based 2-DE of Aβ peptides andre-analyzed published data sets. Our methods showed higher precision and accuracy than other approaches when applied to exposure time series and standard gels. Conclusion Compound fitting as a quantification method for 2-DE spots shows several advantages over other approaches and could be combined with various spot detection methods. The algorithm was scripted in MATLAB (Mathworks) and is available as a supplemental file. PMID:24915860

Direct and transdentinal (indirect) antibacterial activity of commercially available dental gel formulations against Streptococcus mutans.

PubMed

Tüzüner, Tamer; Ulusoy, Ayça Tuba; Baygin, Ozgul; Yahyaoglu, Gorkem; Yalcin, Ilkay; Buruk, Kurtulus; Nicholson, John

2013-01-01

To evaluate the direct and transdentinal (indirect) agar diffusion antibacterial activity of different commercially available antibacterial dental gel formulations against Streptococcus mutans. The commercially available dental gel formulations were Corsodyl® (COG, 1% chlorhexidine), Cervitec® (CEG, 0.2% chlorhexidine + 0.2% sodium fluoride), Forever Bright® (FOB, aloe vera), Gengigel® (GEG, 0.2% hyaluronic acid), 35% phosphoric acid gel and distilled water (control). Direct agar diffusion was performed by isolating three wells from brain-heart infusion agar plates using sterile glass pipettes attached to a vacuum pump and adding 0.1 ml of the gels to each well. Transdentinal (indirect) agar diffusion was performed by applying gel to 0.2- and 0.5-mm-thick human dentin discs previously etched with phosphoric acid and rinsed with distilled water. Zones formed around the wells and the dentin discs were measured and analyzed using Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (p < 0.01). Direct agar diffusion tests showed significant differences among all gel formulations (p < 0.01) except for COG and CEG (p > 0.01). COG and CEG exhibited higher antibacterial effects compared to FOB and GEG (p < 0.01) in both direct and transdentinal (indirect) testing procedures. GEG did not show any antimicrobial activity in transdentinal (indirect) testing. Commercially available dental gels inhibited S. mutans, which may indicate their potential as cavity disinfectants. Copyright © 2013 S. Karger AG, Basel.

Microdisc gel electrophoresis in sodium dodecyl sulfate of organic material from rat otoconial complexes

NASA Technical Reports Server (NTRS)

Ross, M. D.; Pote, K. G.; Rarey, K. E.; Verma, L. M.

1981-01-01

The gravity receptors of all vertebrates utilize a 'test mass' consisting of a complex arrangement of mineral and organic substance that lies over the sensory receptor areas. In most vertebrates, the mineral is a polymorph of calcium carbonate in the form of minute, single crystals called otoconia. An investigation is conducted to determine the number of proteins in otoconial complexes and their molecular weights. The investigation makes use of a microdisk gel electrophoresis method reported by Gainer (1971). The most important finding of the reported research is that analysis of the proteins of the organic material of the otoconial complexes is possible when sensitive microanalytical methods are employed. Further modification of the basic technique employed and the inclusion of other sensitive staining methods should mean that, in the future, protein separation by molecular weight will be possible in sample pools containing only two otoconial masses.

Experimental hut evaluation of linalool spatial repellent agar gel against Anopheles gambiae sensu stricto mosquitoes in a semi-field system in Bagamoyo, Tanzania.

PubMed

Tambwe, Mgeni Mohamed; Mbeyela, Edgar Mtaki; Massinda, Brian Migamyo; Moore, Sarah Jane; Maia, Marta Ferreira

2014-12-05

Malaria vector control is in need of new tools to face its current challenges such as the spread of pyrethroid-resistance and the increase of outdoor feeding mosquitoes. New strategies such as spatial repellents need to be evaluated as supplemental tools to existing control measures such as insecticide treated bed nets and indoor residual spraying. Linalool is a naturally occurring terpene alcohol commonly found in flowers and spices with reportedly repellent properties. Four experimental huts fitted with exit traps and enclosed inside a large screened semi-field system were used for the evaluation. The tested spatial repellent product consisted of an agar gel emanator containing 73% linalool. Two rounds of experiments using a Latin square design were conducted to evaluate the efficacy of the linalool emanators compared to no treatment (negative control) and a transfluthrin coil (positive) against lab-reared disease free Anopheles gambiae s.s.. The emanators were hung inside experimental huts where two volunteers were sleeping unprotected. The outcome measures were repellency, % feeding inhibition, %mortality and post 24 h % mortality. Unlike the mosquito coil, the linalool emanators did not show any feeding inhibition, repellency or induced mortality compared to the negative control. On the other hand mosquitoes kept for 24 h post exposure were 3 times more likely to die after being exposed to two 73% linalool emanators than the negative control. Our results indicate that linalool agar gel emanators are not adequate as a spatial repellent against Anopheles gambiae s.s.. However adding linalool to known repellent formulations could be advantageous, not only because of its pleasant scent but also because of the delayed mortality effect it has on mosquitoes.

2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

PubMed

Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

2009-07-01

The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

Molecular-sieve chromatography and electrophoresis in polyacrylamide gels

PubMed Central

Morris, C. J. O. R.; Morris, Peggy

1971-01-01

1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10°C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results. PMID:5135238

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene.

PubMed

Elek, Zsuzsanna; Dénes, Réka; Prokop, Susanne; Somogyi, Anikó; Yowanto, Handy; Luo, Jane; Souquet, Manfred; Guttman, András; Rónai, Zsolt

2016-09-01

The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

PubMed

Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

2016-09-01

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

PubMed Central

Skinner, Owen S.; Do Vale, Luis H. F.; Catherman, Adam D.; Havugimana, Pierre C.; Valle de Sousa, Marcelo; Domont, Gilberto B.; Kelleher, Neil L.; Compton, Philip D.

2016-01-01

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses. PMID:26967310

Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, P.K.; Lee, Cheng S.; King, J.A.

1997-12-31

The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresismore » (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.« less

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

PubMed

Miller, M J; Maher, V M; McCormick, J J

1992-11-01

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Gel electrophoresis of linear and star-branched DNA

NASA Astrophysics Data System (ADS)

Lau, Henry W.; Archer, Lynden A.

2011-12-01

The electrophoretic mobility of double-stranded DNA in polyacrylamide gel is investigated using an activated hopping model for the transport of a charged object within a heterogeneous medium. The model is premised upon a representation of the DNA path through the gel matrix as a series of traps with alternating large and small cross sections. Calculations of the trap dimensions from gel data show that the path imposes varying degrees of confinement upon migrating analytes, which retard their forward motion in a size-dependent manner. An expression derived for DNA mobility is shown to provide accurate predictions for the dynamics of linear DNA (67-622 bp) in gels of multiple concentrations. For star-branched DNA, the incorporation within the model of a length scale previously proposed to account for analyte architecture [Yuan , Anal. Chem.ANCHAM0003-270010.1021/ac060414w 78, 6179 (2006)] leads to mobility predictions that compare well with experimental results for a wide range of DNA shapes and molecular weights.

Comparison of Pulsed-Field Gel Electrophoresis, Ribotyping, and Plasmid Profiling for Typing of Vibrio anguillarum Serovar O1

PubMed Central

Skov, M. N.; Pedersen, K.; Larsen, J. L.

1995-01-01

A total of 75 Vibrio anguillarum serogroup O1 strains were studied with respect to their plasmid contents, ribotypes, and pulsed-field gel electrophoresis (PFGE) patterns. Eight plasmid profiles and six ribotypes were demonstrated, and one profile was dominant by both typing methods. In contrast, PFGE had very high discriminatory power, demonstrating 35 profiles. On the basis of PFGE patterns, a similarity matrix and a dendrogram were constructed. The results indicated that Scandinavian strains and southern European isolates (with some exceptions) belong to two different clonal lineages. A few strains from the United States and United Kingdom deviated considerably from each other and from Scandinavian and southern European strains. PMID:16535003

21 CFR 866.4900 - Support gel.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Support gel. 866.4900 Section 866.4900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES...) Identification. A support gel for clinical use is a device that consists of an agar or agarose preparation that...

21 CFR 866.4900 - Support gel.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Support gel. 866.4900 Section 866.4900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES...) Identification. A support gel for clinical use is a device that consists of an agar or agarose preparation that...

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale.

PubMed

Gutzweiler, Ludwig; Gleichmann, Tobias; Tanguy, Laurent; Koltay, Peter; Zengerle, Roland; Riegger, Lutz

2017-07-01

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on-demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary- and chip-based methods offering low consumable costs (<0.1$) circumventing cost-intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2-4 μL in volume, a semi-contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non-contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR-grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi-circular cross section of adaptable width (∼200-600 μm) and height (∼30-80 μm) as well as a typical length of 15-55 mm. Layout of such liquid structures is adaptable on-demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label-free DNA ladder (100-1000 bp) at 100 V/cm via in-line staining and laser induced fluorescent end-point detection using an automated prototype. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

PubMed Central

Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

2012-01-01

In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

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…

21 CFR 184.1115 - Agar-agar.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Agar-agar. 184.1115 Section 184.1115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

21 CFR 184.1115 - Agar-agar.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Agar-agar. 184.1115 Section 184.1115 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific...

A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

PubMed Central

Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

2017-01-01

Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples. PMID:28955365

A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches.

PubMed

Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

2017-01-01

Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight : We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

PubMed

Sheen, Hyukho

2016-04-01

Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of internal structure of hydrated agar and gelatin matrices by cryo-SEM.

PubMed

Rahbani, Janane; Behzad, Ali R; Khashab, Niveen M; Al-Ghoul, Mazen

2013-02-01

There has been a considerable interest in recent years in developing polymer gel matrices for many important applications such as 2DE for quantization and separation of a variety of proteins and drug delivery system to control the release of active agents. However, a well-defined knowledge of the ultrastructures of the gels has been elusive. In this study, we report the characterization of two different polymers used in 2DE: Gelatin, a naturally occurring polymer derived from collagen (protein) and agar, a polymer of polysaccharide (sugar) origin. Low-temperature SEM is used to examine the internal structure of these gels in their frozen natural hydrated states. Results of this study show that both polymers have an array of hollow cells that resembles honeycomb structures. While agar pores are almost circular, the corresponding Gaussian curve is very broad exhibiting a range of radii from nearly 370 to 700 nm. Gelatin pores are smaller and more homogeneous reflecting a narrower distribution from nearly 320 to 650 nm. Overall, these ultrastructural findings could be used to correlate with functions of the polymers. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells.

PubMed

Sinha, P; Hütter, G; Köttgen, E; Dietel, M; Schadendorf, D; Lage, H

1998-11-18

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

Introducing basic molecular biology to Turkish rural and urban primary school children via hands-on PCR and gel electrophoresis activities.

PubMed

Selli, Cigdem; Yıldırım, Gokce; Kaymak, Aysegul; Karacicek, Bilge; Ogut, Deniz; Gungor, Turkan; Erem, Erdem; Ege, Mehmet; Bümen, Nilay; Tosun, Metiner

2014-01-01

This study includes the results of a 2-day education project titled "Molecular Biology Laboratory Summer School, MoBiLYO." The project was held at a University Research Center by scientists from Department of Pharmacology and graduate students. The project was composed of introductory lectures, model construction, DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis. The participants were 13-year-old eighth-graders attending primary schools affiliated with Ministry of National Education in urban and rural areas of Izmir, Turkey. The purpose of this study was to introduce basic molecular biology concepts through individually performed experiments such as PCR and gel electrophoresis integrated with creative drama. The students were assessed at the beginning and the end of each project day via mini-tests, experimental and presentation skills evaluation forms. Data showed that students' knowledge about DNA structure and basic molecular biology techniques significantly increased. On the basis of experimental and presentational skills, there was no significant difference between kids from urban and rural schools or between public and boarding public schools, whereas the average score of girls was significantly higher than that of boys. In conclusion, individually performed experiments integrated with creative drama significantly increased students' perception of complex experimental procedures on basic molecular biology concepts. Data suggests that integration of these concepts into the science and technology curriculum of Turkish primary education may support the recruitment of future scientists who can handle rapidly developing genomic techniques that will affect our everyday life. © 2014 by The International Union of Biochemistry and Molecular Biology.

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.

Nonlinear effects on electrophoresis of a charged dielectric nanoparticle in a charged hydrogel medium

NASA Astrophysics Data System (ADS)

Bhattacharyya, S.; De, Simanta

2016-09-01

The impact of the solid polarization of a charged dielectric particle in gel electrophoresis is studied without imposing a weak-field or a thin Debye length assumption. The electric polarization of a dielectric particle due to an external electric field creates a non-uniform surface charge density, which in turn creates a non-uniform Debye layer at the solid-gel interface. The solid polarization of the particle, the polarization of the double layer, and the electro-osmosis of mobile ions within the hydrogel medium create a nonlinear effect on the electrophoresis. We have incorporated those nonlinear effects by considering the electrokinetics governed by the Stokes-Brinkman-Nernst-Planck-Poisson equations. We have computed the governing nonlinear coupled set of equations numerically by adopting a finite volume based iterative algorithm. Our numerical method is tested for accuracy by comparing with several existing results on free-solution electrophoresis as well as results based on the Debye-Hückel approximation. Our computed result shows that the electrophoretic velocity decreases with the rise of the particle dielectric permittivity constant and attains a saturation limit at large values of permittivity. A significant impact of the solid polarization is found in gel electrophoresis compared to the free-solution electrophoresis.

Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

PubMed

Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

2011-03-01

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

Optimization of pectinase immobilization on grafted alginate-agar gel beads by 24 full factorial CCD and thermodynamic profiling for evaluating of operational covalent immobilization.

PubMed

Abdel Wahab, Walaa A; Karam, Eman A; Hassan, Mohamed E; Kansoh, Amany L; Esawy, Mona A; Awad, Ghada E A

2018-07-01

Pectinase produced by a honey derived from the fungus Aspergillus awamori KX943614 was covalently immobilized onto gel beads made of alginate and agar. Polyethyleneimine, glutaraldehyde, loading time and enzyme's units were optimized by 2 4 full factorial central composite design (CCD). The immobilization process increased the optimal working pH for the free pectinase from 5 to a broader range of pH4.5-5.5 and the optimum operational temperature from 55°C to a higher temperature, of 60°C, which is favored to reduce the enzyme's microbial contamination. The thermodynamics studies showed a thermal stability enhancement against high temperature for the immobilized formula. Moreover, an increase in half-lives and D-values was achieved. The thermodynamic studies proved that immobilization of pectinase made a remarkable increase in enthalpy and free energy because of enzyme stability enhancement. The reusability test revealed that 60% of pectinase's original activity was retained after 8 successive cycles. This gel formula may be convenient for immobilization of other industrial enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.

PubMed

Martin, D S; Desser, S S; Hong, H

1992-04-01

Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences.

Microbial Community Structure of Korean Cabbage Kimchi and Ingredients with Denaturing Gradient Gel Electrophoresis.

PubMed

Hong, Sung Wook; Choi, Yun-Jeong; Lee, Hae-Won; Yang, Ji-Hee; Lee, Mi-Ai

2016-06-28

Kimchi is a traditional Korean fermented vegetable food, the production of which involves brining of Korean cabbage, blending with various other ingredients (red pepper powder, garlic, ginger, salt-pickled seafood, etc.), and fermentation. Recently, kimchi has also become popular in the Western world because of its unique taste and beneficial properties such as antioxidant and antimutagenic activities, which are derived from the various raw materials and secondary metabolites of the fermentative microorganisms used during production. Despite these useful activities, analysis of the microbial community present in kimchi has received relatively little attention. The objective of this study was to evaluate the bacterial community structure from the raw materials, additives, and final kimchi product using the culture-independent method. Specifically, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the 16S rRNA partial sequences of the microflora. One primer set for bacteria, 341F(GC)-518R, reliably produced amplicons from kimchi and its raw materials, and these bands were clearly separated on a 35-65% denaturing gradient gel. Overall, 117 16S rRNA fragments were identified by PCR-DGGE analysis. Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc gelidum, and Leuconostoc mesenteroides were the dominant bacteria in kimchi. The other strains identified were Tetragenococcus, Pseudomonas, Weissella, and uncultured bacterium. Comprehensive analysis of these microorganisms could provide a more detailed understanding of the biologically active components of kimchi and help improve its quality. PCR-DGGE analysis can be successfully applied to a fermented food to detect unculturable or other species.

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis.

PubMed

Mineyama, R; Yoshino, S; Maeda, N

2007-01-01

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.

Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

PubMed

Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

2016-02-01

Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE).

PubMed

Lam, V M; Huang, W; Lam, S T; Yeung, C Y; Johnson, P H

1996-03-01

We describe here the use of denaturing gradient gel electrophoresis (DGGE) to detect the most common Chinese glucose-6-phosphate dehydrogenase (G6PD) variants, which are the single point mutations: G-->T at nt 1376, G-->A at 1388 both in exon 12 and A-->G at nt 95 in exon 02. In each case, the mutant allele resolves well from the normal allele(s). The distinct heteroduplex bands are characteristic of a particular genotype suggesting that this feature is very useful for identifying all heterozygous carriers for this and other X-linked diseases. When the analysis is extended to other exons, DGGE scans the gene and coupled with direct sequencing, it leads to the identification of new G6PD variation(s). With this approach, we identified a mutation in exon 9 which had not been reported in Hong Kong. Since DGGE can rapidly screen many unknown samples in one gel, this approach could be used to diagnose these G6PD mutations and to identify the at-risk for counselling.

Data on DNA gel sample load, gel electrophoresis, PCR and cost analysis.

PubMed

Kuhn, Ramona; Böllmann, Jörg; Krahl, Kathrin; Bryant, Isaac Mbir; Martienssen, Marion

2018-02-01

The data presented in this article provide supporting information to the related research article "Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples" (Kuhn et al., 2017) [1]. In that article, we compared the suitability of ten selected DNA extraction methods based on DNA quality, purity, quantity and applicability to universal PCR. Here we provide the data on the specific DNA gel sample load, all unreported gel images of crude DNA and PCR results, and the complete cost analysis for all tested extraction procedures and in addition two commercial DNA extraction kits for soil and water.

Morphological identification of Candida species on glucose agar, rice extract agar and corn meal agar with and without Tween-80.

PubMed

Joshi, K R; Solanki, A; Prakash, P

1993-01-01

A comparative study for the identification of 32 known strains of Candida species on the basis of morphology on glucose agar, rice extract agar and corn meal agar with and without Tween 80 revealed that when Tween 80 is incorporated in the media identification is possible for 96.8% of the species within 48 hours on rice extract agar and for 96.8% of the species within 48 hours on rice extract agar and for 90.6% of the species on glucose agar. The germ tubes and chlamydospores were also produced more on rice extract agar than on 0.1% glucose agar. Rice extract agar with Tween 80 can be used as single medium for morphologic identification of Candida species. The inoculated medium is first incubated at 37 degrees C for 3 hours and examined for germ tube formation and then incubated at 25 degrees C for 24 to 72 hours and examined for appearance of chlamydospores and mycelial morphology.

[A structural protein study of the influenza A (H1N1) virus by polyacrylamide gel electrophoresis].

PubMed

Pérez Guevara, M T; Savón Valdés, C; Rivas Arjona, M; Goyenechea Hernández, A

1992-01-01

Influenza is an acute respiratory disease typically appearing as an epidemic. Three immunological types of the influenza virus are known: A, B and C. Continually, antigen changes occur, especially in type A. Therefore, a comparative study was carried out on 4 influenza A(H1N1) virus strains in relation to protein structure (surface antigens), by using polyacrylamide gel electrophoresis by the modified Laemmli method. The objective was to compare the structural proteins of the A/Havana/1292/78 (H1N1) national strain with the proteins of 3 international pattern strains. In all the cases, 6 bands were detected by densitometry. In the 4 strains studied the most abundant protein was M. Great differences between the Cuban strain and the 3 international patterns were not seen.

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes.

PubMed

Singh, R; Chénier, D; Bériault, R; Mailloux, R; Hamel, R D; Appanna, V D

2005-09-30

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

PubMed Central

Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

2009-01-01

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

Preprocessing of 2-Dimensional Gel Electrophoresis Images Applied to Proteomic Analysis: A Review.

PubMed

Goez, Manuel Mauricio; Torres-Madroñero, Maria Constanza; Röthlisberger, Sarah; Delgado-Trejos, Edilson

2018-02-01

Various methods and specialized software programs are available for processing two-dimensional gel electrophoresis (2-DGE) images. However, due to the anomalies present in these images, a reliable, automated, and highly reproducible system for 2-DGE image analysis has still not been achieved. The most common anomalies found in 2-DGE images include vertical and horizontal streaking, fuzzy spots, and background noise, which greatly complicate computational analysis. In this paper, we review the preprocessing techniques applied to 2-DGE images for noise reduction, intensity normalization, and background correction. We also present a quantitative comparison of non-linear filtering techniques applied to synthetic gel images, through analyzing the performance of the filters under specific conditions. Synthetic proteins were modeled into a two-dimensional Gaussian distribution with adjustable parameters for changing the size, intensity, and degradation. Three types of noise were added to the images: Gaussian, Rayleigh, and exponential, with signal-to-noise ratios (SNRs) ranging 8-20 decibels (dB). We compared the performance of wavelet, contourlet, total variation (TV), and wavelet-total variation (WTTV) techniques using parameters SNR and spot efficiency. In terms of spot efficiency, contourlet and TV were more sensitive to noise than wavelet and WTTV. Wavelet worked the best for images with SNR ranging 10-20 dB, whereas WTTV performed better with high noise levels. Wavelet also presented the best performance with any level of Gaussian noise and low levels (20-14 dB) of Rayleigh and exponential noise in terms of SNR. Finally, the performance of the non-linear filtering techniques was evaluated using a real 2-DGE image with previously identified proteins marked. Wavelet achieved the best detection rate for the real image. Copyright © 2018 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Production and hosting by Elsevier B

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis.

PubMed

Lindahl, Susanne; Söderlund, Robert; Frosth, Sara; Pringle, John; Båverud, Viveca; Aspán, Anna

2011-11-21

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers

PubMed Central

Walter, J.; Tannock, G. W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Loach, D. M.; Munro, K.; Alatossava, T.

2000-01-01

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. PMID:10618239

Attempt to run urinary protein electrophoresis using capillary technique.

PubMed

Falcone, Michele

2014-10-01

The study of urinary protein has a predominant place in the diagnosis of kidney disease. The most common technique is agarose gel electrophoresis (AGE). For several years, the technique of choice applied to the analysis of serum proteins has been CE, a system that uses capillary fused silica, subjected to high voltage to separate and measure serum proteins. The purpose of this paper was to perform capillary electrophoresis on urinary proteins which, at present, are not interpretable due to the many nonspecific peaks visible when using gel electrophoresis. In order to carry out our research, we used a capillary V8 analyzer together with an agarose gel system from the same company. AGE was taken as the reference method, for which urine was used without any pretreatment. For the V8 system, urine was subjected to purification on granular-activated carbon and then inserted into the V8 analyzer, selecting a program suitable for liquids with low protein content. We examined 19 urine samples collected over 24 hrs from both hospitalized and external patients with different types of proteinuria plus a serum diluted 1/61 considered as a control to recognize the bands. Both methods showed the same protein fractions and classified the proteinuria in a similar way. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Running DNA Mini-Gels in 20 Minutes or Less Using Sodium Boric Acid Buffer

ERIC Educational Resources Information Center

Jenkins, Kristin P.; Bielec, Barbara

2006-01-01

Providing a biotechnology experience for students can be challenging on several levels, and time is a real constraint for many experiments. Many DNA based methods require a gel electrophoresis step, and although some biotechnology procedures have convenient break points, gel electrophoresis does not. In addition to the time required for loading…

Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.

PubMed

Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M

2011-08-01

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans.

PubMed

Boriollo, Marcelo Fabiano Gomes; Dias, Ricardo Antunes; Fiorini, João Evangelista; Oliveira, Nelma de Mello Silva; Spolidório, Denise Madalena Palomari; de Souza, Henrique Marques Barbosa; Figueira, Antonio Vargas de Oliveira; Pizzirani-Kleiner, Aline Aparecida

2010-09-01

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE)xS(SM)(EK)xS(SM)(SSRs)). Clustering analyses showed a mean of 9+/-12.4 isolates per cluster (3.8+/-8 isolates/taxon) for MLEE, 6.2+/-4.9 isolates per cluster (4+/-4.5 isolates/taxon) for SSRs, and 4.1+/-2.3 isolates per cluster (2.6+/-2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S(J)) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented

The Make 2D-DB II package: conversion of federated two-dimensional gel electrophoresis databases into a relational format and interconnection of distributed databases.

PubMed

Mostaguir, Khaled; Hoogland, Christine; Binz, Pierre-Alain; Appel, Ron D

2003-08-01

The Make 2D-DB tool has been previously developed to help build federated two-dimensional gel electrophoresis (2-DE) databases on one's own web site. The purpose of our work is to extend the strength of the first package and to build a more efficient environment. Such an environment should be able to fulfill the different needs and requirements arising from both the growing use of 2-DE techniques and the increasing amount of distributed experimental data.

Differential Single Nucleotide Polymorphism-Based Analysis of an Outbreak Caused by Salmonella enterica Serovar Manhattan Reveals Epidemiological Details Missed by Standard Pulsed-Field Gel Electrophoresis

PubMed Central

Scaltriti, Erika; Sassera, Davide; Comandatore, Francesco; Morganti, Marina; Mandalari, Carmen; Gaiarsa, Stefano; Bandi, Claudio; Zehender, Gianguglielmo; Bolzoni, Luca; Casadei, Gabriele

2015-01-01

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n = 15) and food, feed, animal, and environmental sources (n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system. PMID:25653407

Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis

PubMed Central

Pooideh, Mohammad; Jabbarzadeh, Ismail; Ranjbar, Reza; Saifi, Mahnaz

2015-01-01

Background: Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used. Objectives: During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method. Materials and Methods: Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software. Results: Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed. Conclusions: Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly

Recolonization of mutans Streptococci after application of chlorhexidine gel.

PubMed

Vale, Glauber Campos; Cury, Altair Antoninha Del Bel; Arthur, Rodrigo Alex; Cury, Jaime Aparecido; Tabchoury, Cínthia Pereira Machado

2014-01-01

Streptococcus mutans is specifically suppressed by intensive treatment with chlorhexidine gel, but the time for recolonization and the effect on other oral bacteria are not totally clear. In this study, recolonization of mutans streptococci was evaluated in nine healthy adult volunteers, who were highly colonized with this microorganism. Stimulated saliva was collected before (baseline) and at 1, 7, 14, 21 and 28 days after application of 1% chlorhexidine gel on volunteers' teeth for two consecutive days. On each day, the gel was applied using disposable trays for 3 x 5 min with intervals of 5 min between each application. Saliva was plated on blood agar to determine total microorganisms (TM); on mitis salivarius agar to determine total streptococci (TS) and on mitis salivarius agar plus bacitracin to determine mutans streptococci (MS). Chlorhexidine was capable of reducing the counts of MS and the proportion of MS with regard to total microorganisms (%MS/TM) (p<0.05), but these values did not differ statistically from baseline (p>0.05) after 14 days for MS and 21 days for %MS/TM. The counts of TM and TS and the proportion of MS to total streptococci did not differ statistically from baseline (p>0.05) after chlorhexidine treatment. The results suggest that the effect of chlorhexidine gel treatment on suppression of mutans streptococci is limited to less than a month in highly colonized individuals.

Automated image alignment for 2D gel electrophoresis in a high-throughput proteomics pipeline.

PubMed

Dowsey, Andrew W; Dunn, Michael J; Yang, Guang-Zhong

2008-04-01

The quest for high-throughput proteomics has revealed a number of challenges in recent years. Whilst substantial improvements in automated protein separation with liquid chromatography and mass spectrometry (LC/MS), aka 'shotgun' proteomics, have been achieved, large-scale open initiatives such as the Human Proteome Organization (HUPO) Brain Proteome Project have shown that maximal proteome coverage is only possible when LC/MS is complemented by 2D gel electrophoresis (2-DE) studies. Moreover, both separation methods require automated alignment and differential analysis to relieve the bioinformatics bottleneck and so make high-throughput protein biomarker discovery a reality. The purpose of this article is to describe a fully automatic image alignment framework for the integration of 2-DE into a high-throughput differential expression proteomics pipeline. The proposed method is based on robust automated image normalization (RAIN) to circumvent the drawbacks of traditional approaches. These use symbolic representation at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in modelling and alignment. In RAIN, a third-order volume-invariant B-spline model is incorporated into a multi-resolution schema to correct for geometric and expression inhomogeneity at multiple scales. The normalized images can then be compared directly in the image domain for quantitative differential analysis. Through evaluation against an existing state-of-the-art method on real and synthetically warped 2D gels, the proposed analysis framework demonstrates substantial improvements in matching accuracy and differential sensitivity. High-throughput analysis is established through an accelerated GPGPU (general purpose computation on graphics cards) implementation. Supplementary material, software and images used in the validation are available at http://www.proteomegrid.org/rain/.

Preparation of amine-impregnated silica foams using agar as the gelling agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jardim, Iara M., E-mail: iaramj01@yahoo.com.br

In this work we successfully prepared amine-impregnated gel-cast silica foams using agar and atmospheric air as the gelling agent and heat treatment atmosphere, respectively. The concentration of 3,6-anhydrogalactose in agar was evaluated by ultraviolet–visible spectroscopy (UV–Vis). The obtained foams were examined by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG) coupled to mass spectrometry (TG-MS), scanning electron microscopy (SEM), X-ray microtomography (micro-CT), and Archimedes method. The cold crushing strength of the materials prepared in this work was assessed using a mechanical testing stage available in the micro-CT system. The obtained foams exhibited a highly interconnected pore network, with an expressivemore » presence of open pores. Samples heat-treated at 1300 °C for 2 h showed both an expressive porosity (≈ 77%) and a significant cold crushing strength (≈ 1.4 MPa). It was observed that the calcination of the prepared materials at 1200 °C for times as long as 16 h may lead to the rupture of pore walls. FTIR and TG-MS revealed that amine groups were properly incorporated into the foams structure. - Highlights: •Successful preparation of amine-impregnated gel-cast silica foams •Agar used as the gelling agent •Samples with expressive porosity and cold crushing strength •Sintering times as long as 16 h led to the rupture of the pore network.« less

Probing Biomolecular Structures and Dynamics of Single Molecules Using In-Gel Alternating-Laser Excitation

PubMed Central

Santoso, Yusdi; Kapanidis, Achillefs N.

2009-01-01

Gel electrophoresis is a standard biochemical technique used for separating biomolecules on the basis of size and charge. Despite the use of gels in early single-molecule experiments, gel electrophoresis has not been widely adopted for single-molecule fluorescence spectroscopy. We present a novel method that combines gel electrophoresis and single-molecule fluorescence spectroscopy to simultaneously purify and analyze biomolecules in a gel matrix. Our method, in-gel ALEX, uses non-denaturing gels to purify biomolecular complexes of interest from free components, aggregates, and non-specific complexes. The gel matrix also slows down translational diffusion of molecules, giving rise to long, high-resolution time traces without surface immobilization, which allow extended observations of conformational dynamics in a biologically friendly environment. We demonstrated the compatibility of this method with different types of single molecule spectroscopy techniques, including confocal detection and fluorescence-correlation spectroscopy. We demonstrated that in-gel ALEX can be used to study conformational dynamics at the millisecond timescale; by studying a DNA hairpin in gels, we directly observed fluorescence fluctuations due to conformational interconversion between folded and unfolded states. Our method is amenable to the addition of small molecules that can alter the equilibrium and dynamic properties of the system. In-gel ALEX will be a versatile tool for studying structures and dynamics of complex biomolecules and their assemblies. PMID:19863108

Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

2013-12-19

To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Epidemiologic study of Taylorella equigenitalis strains by field inversion gel electrophoresis of genomic restriction endonuclease fragments.

PubMed Central

Bleumink-Pluym, N; ter Laak, E A; van der Zeijst, B A

1990-01-01

Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration. Images PMID:2172296

Effect of sieving polymer concentration on separation of 100 bp DNA Ladder by capillary gel electrophoresis

NASA Astrophysics Data System (ADS)

Nakazumi, T.; Hara, Y.

2017-09-01

We studied the effect of sieving polymer concentration on separation of a 100 bp DNA Ladder by capillary gel electrophoresis (CGE) using hydroxyethyl cellulose (HEC) with a molecular size of 1000 k. For measurement purposes, we selected a fused silica capillary with total length of 15 cm and effective length of 7.5 cm; this was applied to compact CGE equipment for a Point-Care-Testing (POCT) system. Measurement results of the 100 bp DNA Ladder sample indicated that small DNA separation was significantly affected by HEC sieving polymer concentration. This was due to the level of entanglement between small DNA molecules and the sieving polymer chain significantly influencing migration time, mobility, and resolution length of the CGE process. We concluded that 1.0 w/v % HEC sieving polymer concentration was optimal for CGE separation of DNA ≥1000bp in the 100 bp DNA Ladder (100-1500 bp) when using the short-length capillary.

Analysis of electrophoresis performance

NASA Technical Reports Server (NTRS)

Roberts, G. O.

1984-01-01

The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

PubMed Central

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

1999-01-01

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers.

PubMed

Hanna-Addams, Sarah; Wang, Zhigao

2018-04-26

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Imaging of Selenium by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) in 2-D Electrophoresis Gels and Biological Tissues.

PubMed

Cruz, Elisa Castañeda Santa; Susanne Becker, J; Sabine Becker, J; Sussulini, Alessandra

2018-01-01

Selenium and selenoproteins are important components of living organisms that play a role in different biological processes. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a powerful analytical technique that has been employed to obtain distribution maps of selenium in biological tissues in a direct manner, as well as in selenoproteins, previously separated by their molecular masses and isoelectric points using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In this chapter, we present the protocols to perform LA-ICP-MS imaging experiments, allowing the distribution visualization and determination of selenium and/or selenoproteins in biological systems.

Comparison of arbitrarily primed PCR and macrorestriction (pulsed-field gel electrophoresis) typing of Pseudomonas aeruginosa strains from cystic fibrosis patients.

PubMed Central

Kersulyte, D; Struelens, M J; Deplano, A; Berg, D E

1995-01-01

Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency. PMID:7559985

Investigating Freshwater Periphyton Community Response to Uranium with Phospholipid Fatty Acid and Denaturing Gradient Gel Electrophoresis Analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Jack A.; Bunn, Amoret L.; McKinstry, Craig A.

2008-04-01

Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure and genetic diversity were used to investigate impacts of uranium exposure on periphyton. Laboratory exposures of periphyton in river water amended with uranium were performed for 5 days, followed by 2 days of uranium depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 µg L-1 uranium. Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns found no changes in community or genetic structure relatedmore » to uranium exposure. We suggest that the periphyton community as a whole is not impacted by exposures of uranium up to a dose of 100 µg L-1. These findings have significance for the assessment and prediction of uranium impacts on aquatic ecosystems.« less

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

PubMed

Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher

2012-04-01

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

Differential single nucleotide polymorphism-based analysis of an outbreak caused by Salmonella enterica serovar Manhattan reveals epidemiological details missed by standard pulsed-field gel electrophoresis.

PubMed

Scaltriti, Erika; Sassera, Davide; Comandatore, Francesco; Morganti, Marina; Mandalari, Carmen; Gaiarsa, Stefano; Bandi, Claudio; Zehender, Gianguglielmo; Bolzoni, Luca; Casadei, Gabriele; Pongolini, Stefano

2015-04-01

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n=15) and food, feed, animal, and environmental sources (n=24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA Sequencing by Capillary Electrophoresis

PubMed Central

Karger, Barry L.; Guttman, Andras

2009-01-01

Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

PubMed

Bjerneld, Erik J; Johansson, Johan D; Laurin, Ylva; Hagner-McWhirter, Åsa; Rönn, Ola; Karlsson, Robert

2015-09-01

A pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range-more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250pmol in 20μl using a Tris-HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS-PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipoprotein Agarose Gel Electrophoresis. Application in HDL-Cholesterol Methodology.

DTIC Science & Technology

1985-06-01

on determination of high-density lipo- protein cholesterol by precipitation with sodium phosphotungstate- magnesium. Clin Chem 25(4):560 (1979). 6...determinations of cholesterol levels in supernates obtained after addition of various amounts of precipitants , lipo- protein electrophoresis can help to...plotted against the corresponding volumes of sodium phosphotungstate-MgCl2 (NAPT) precipitant added. 10 ’ ’’ ’i - n

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

USDA-ARS?s Scientific Manuscript database

Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transfe...

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network.

PubMed

Li, Yan; Buch, Jesse S; Rosenberger, Frederick; DeVoe, Don L; Lee, Cheng S

2004-02-01

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.

Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

PubMed

Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

2014-07-04

Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

A study of electrochemical devices based on Agar-Agar-NH4I biopolymer electrolytes

NASA Astrophysics Data System (ADS)

Selvalakshmi, S.; Mathavan, T.; Selvasekarapandian, S.; Premalatha, M.

2018-04-01

A polymer electrolyte system has been developed using a biopolymer namely, Agar-Agar in combination with ammonium iodide in different weight percentages by solution casting technique. The films were characterized electrically by AC Impedance Spectroscopy for its conductivity. The highest conductivity achieved at room temperature was for 50 wt. % agar-agar: 50 wt. % NH4I with a conductivity value of 1.20 × 10-4 Scm-1. An electrochemical cell was fabricated in the configuration of: Zn + ZnSO4.7H2O + graphite (anode) | 50 wt. % (Agar-agar): 50 wt. % NH4I (electrolyte) | PbO2 + V2O5 + graphite (cathode) and it produced a maximum open circuit voltage of 1.73 V. A single PEM fuel cell was constructed with the highest conducting sample (50 wt. % (Agar-agar): 50 wt. % NH4I) and it exhibited an output voltage of 408mV.

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque.

PubMed

Fujimoto, C; Maeda, H; Kokeguchi, S; Takashiba, S; Nishimura, F; Arai, H; Fukui, K; Murayama, Y

2003-08-01

Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.

Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity.

PubMed

Kirzek, D M; Rick, M E

2001-03-01

A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.

Characterization of Trichuris skrjabini by isoenzyme gel electrophoresis: comparative study with Trichuris ovis.

PubMed

Cutillas, C; German, P; Arias, P; Guevara, D

1996-10-01

Morphological and biometric studies were performed in Trichuris skrjabini (Baskakov, 1924) collected from the caecum of Capra hircus. The LDH (EC 1.1.1.27.), G6PD (EC 1.1.1.49.), GPI (EC 5.3.1.9.), MDH (EC 1.1.1.37) and malic enzyme (ME) (EC 1.1.1.40) isoenzymatic patterns of T. skrjabini were determined by starch gel electrophoresis. The G6PD and GPI isoenzymatic patterns of T. skrjabini displayed two anodic bands for both enzymes: one fast migration band and one band near the origin. This isoenzymatic pattern was interpreted as two gene loci encoding both enzymes. The LDH isoenzymatic pattern of T. skrjabini was characterized by the presence of a cathodically migrating band, while the MDH isoenzymatic pattern showed a very slow cathodic band. These two phenotypes were interpreted as the expression of a homozygous state of a gene locus for LDH and MDH in T. skrjabini. The ME isoenzymatic pattern was characterized by the presence of a single anodic band. Further, comparative isoenzymatic studies were carried out between T. skrjabini and T. ovis. The different G6PD, GPI, LDH, MDH and ME isoenzymatic patterns observed for both species allowed us to distinguish them and therefore to use isoenzymatic patterns as a diagnostic tool to differentiate species of Trichuris.

[Single cell gel electrophoresis of a magnesium alloy coated with beta-tricalcium phosphate].

PubMed

Hao, Yu-quan; Tan, Li-li; Yan, Ting-ting; Yan, Xiu-lin; Yang, Ke; Ai, Hong-jun

2009-10-01

To evaluate the genotoxicity of a magnesium alloy coated with beta-tricalcium phosphate (beta-TCP). Four groups were designed. In the first group, AZ31B magnesium alloy surface was coated with beta-TCP using chemical bath deposition, and in the second group magnesium alloy was tested. The other two groups were negative control (pure titanium) and positive control groups (0.5 mg/L bleomycin). Single cell gel electrophoresis was adopted to investigate genotoxicity of the alloy samples in different groups, and 60 cells from each group were analysed. Tail moment and tail DNA percentage were used as reliable indicators to show DNA damage in lymphocytes induced by every testing sample. Student-Newman-Keuls (SNK) test was used to compare results from 4 groups. There were no significant differences in tail moment and tail DNA percentage between magnesium alloy group [(0.52 +/- 0.12), (6.82 +/- 1.81)%] and magnesium alloy coated with beta-TCP group [(0.51 +/- 0.12), (6.89 +/- 1.93)%, P > 0.05]. Tail moment and tail DNA percentage in negative group were (0.47 +/- 0.14) and (6.29 +/- 1.64)%, and tail moment and tail DNA percentage in positive group were (5.17 +/- 1.23) and (22.09 +/- 4.51)%. No significant increase was found in DNA damage in lymphocytes induced by magnesium alloy coated with beta-TCP.

Antibiotic-containing hyaluronic acid gel as an antibacterial carrier: Usefulness of sponge and film-formed HA gel in deep infection.

PubMed

Matsuno, Hiroaki; Yudoh, Kazuo; Hashimoto, Masamichi; Himeda, Yasukazu; Miyoshi, Teruzo; Yoshida, Kaoru; Kano, Syogo

2006-03-01

We have developed a novel bioabsorbable antibacterial carrier using hyaluronic acid (HA) gel for prevention and treatment of orthopedic infections. In this study, we investigated the in vivo antibacterial effects of two forms of this new material, an HA gel sponge and an HA gel film. A titanium cylinder was inserted into the intramedullary cavity of each rabbit femur, along with an HA gel sponge or HA gel film containing antibiotics. The HA gel sponge contained gentamycin, vancomycin, tobramycin, or minomycin. The HA gel film contained gentamycin or vancomycin. After 0, 7, and 14 days, the rabbit bone marrow was collected, and the antibacterial activity of the HA gel was determined by agar diffusion test. As a control, we used Septocoll, a commercially available antibacterial carrier. Both the HA gel sponge and HA gel film exhibited antibacterial activity. The present results indicate that HA gel containing antibiotics is a clinically useful bioabsorbable antibacterial carrier. Copyright 2006 Orthopaedic Research Society.

Capillary Zone Electrophoresis for the Analysis of Peptides: Fostering Students' Problem-Solving and Discovery Learning in an Undergraduate Laboratory Experiment

ERIC Educational Resources Information Center

Albright, Jessica C.; Beussman, Douglas J.

2017-01-01

Capillary electrophoresis is an important analytical separation method used to study a wide variety of samples, including those of biological origin. Capillary electrophoresis may be covered in the classroom, especially in advanced analytical courses, and while many students are exposed to gel electrophoresis in biology or biochemistry…

In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.

PubMed

Bello, F D; Walter, J; Hertel, C; Hammes, W P

2001-07-01

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

Molecular identification of the microbiota of French sourdough using temporal temperature gradient gel electrophoresis.

PubMed

Ferchichi, Mounir; Valcheva, Rosica; Prévost, Hervé; Onno, Bernard; Dousset, Xavier

2007-01-01

The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6-V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroïdes subsp. mesenteroïdes. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.

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

Pulsed-field gel electrophoresis of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections.

PubMed

Hanifah, Y A; Hiramatsu, K

1994-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.

Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis.

PubMed

Miettinen, M K; Björkroth, K J; Korkeala, H J

1999-02-18

One dominating strain of serotype 1/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant. Samples were taken from the production environment, equipment and ice cream during the years 1990-1997. Serotyping divided the isolates into two serovars, 1/2b and 4b. Three rare-cutting enzymes (ApaI, AscI and SmaI) were used in the creation of PFGE patterns. AscI resulted in the best restriction enzyme digestion patterns (REDPs) for visual comparison. Eight different AscI REDPs were obtained, whereas ApaI produced six and SmaI seven banding patterns. When one-band differences are taken into account, 12 different PFGE types were distinguished based on information obtained with all three enzymes. The dominant PFGE type was found to have persisted in the ice cream plant for seven years. Improved and precisely targeted cleaning and disinfection practices combined with structural changes making for easier cleaning of the packaging machine, resulted in eradication of L. monocytogenes from this plant.

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.

Multilocus Sequence Typing Compared to Pulsed-Field Gel Electrophoresis for Molecular Typing of Pseudomonas aeruginosa▿

PubMed Central

Johnson, Jennifer K.; Arduino, Sonia M.; Stine, O. Colin; Johnson, Judith A.; Harris, Anthony D.

2007-01-01

For hospital epidemiologists, determining a system of typing that is discriminatory is essential for measuring the effectiveness of infection control measures. In situations in which the incidence of resistant Pseudomonas aeruginosa is increasing, the ability to discern whether it is due to patient-to-patient transmission versus an increase in patient endogenous strains is often made on the basis of molecular typing. The present study compared the discriminatory abilities of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for 90 P. aeruginosa isolates obtained from cultures of perirectal surveillance swabs from patients in an intensive care unit. PFGE identified 85 distinct types and 76 distinct groups when similarity cutoffs of 100% and 87%, respectively, were used. By comparison, MLST identified 60 sequence types that could be clustered into 11 clonal complexes and 32 singletons. By using the Simpson index of diversity (D), PFGE had a greater discriminatory ability than MLST for P. aeruginosa isolates (D values, 0.999 versus 0.975, respectively). Thus, while MLST was better for detecting genetic relatedness, we determined that PFGE was more discriminatory than MLST for determining genetic differences in P. aeruginosa. PMID:17881548

Exposures of Sus scrofa to a TASER(®) conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

PubMed

Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

2014-12-01

In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30 s exposures of anesthetized pigs (Sus scrofa) to a TASER (®) C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures.

The new horizon in 2D electrophoresis: new technology to increase resolution and sensitivity.

PubMed

Moche, Martin; Albrecht, Dirk; Maaß, Sandra; Hecker, Michael; Westermeier, Reiner; Büttner, Knut

2013-06-01

A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of proteomic differences between liquefied after-cataracts and normal lenses using two-dimensional gel electrophoresis and mass spectrometry

PubMed Central

Ge, Jia-Jia; Huang, Yu-Sen

2017-01-01

AIM To analyze and identify the proteomic differences between liquefied after-cataracts and normal lenses by means of liquefied chromatography-tandem mass spectrometry (LC-MS/MS). METHODS Three normal lenses and three liquefied after-cataracts were exposed to depolymerizing reagents to extract the total proteins. Protein concentrations were separated using two-dimensional gel electrophoresis (2-DE). The digitized images obtained with a GS-800 scanner were then analyzed with PDQuest7.0 software to detect the differentially-expressed protein spots. These protein spots were cut from the gel using a proteome work spot cutter and subjected to in-gel digestion with trypsin. The digested peptide separation was conducted by LC-MS/MS. RESULTS The 2-DE maps showed that lens proteins were in a pH range of 3-10 with a relative molecular weight of 21-70 kD. The relative molecular weight of the more abundant proteins was localized at 25-50 kD, and the isoelectric points were found to lie between PI 4-9. The maps also showed that the protein level within the liquefied after-cataracts was at 29 points and significantly lower than in normal lenses. The 29 points were identified by LC-MS/MS, and ten of these proteins were identified by mass spectrometry and database queries: beta-crystallin B1, glyceraldehyde-3-phosphate dehydrogenase, carbonyl reductase (NADPH) 1, cDNA FLJ55253, gamma-crystallin D, GAS2-like protein 3, sorbitol dehydrogenase, DNA FLJ60282, phosphoglycerate kinase, and filensin. CONCLUSION The level of the ten proteins may play an important role in the development of liquefied after-cataracts. PMID:28944190

DNA ELECTROPHORESIS AT SURFACES

DOE Office of Scientific and Technical Information (OSTI.GOV)

RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

2003-09-01

During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in themore » different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.« less

Bio-barcode gel assay for microRNA

NASA Astrophysics Data System (ADS)

Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

2014-02-01

MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

Direct analysis of in-gel proteins by carbon nanotubes-modified paper spray ambient mass spectrometry.

PubMed

Han, Feifei; Yang, Yuhan; Ouyang, Jin; Na, Na

2015-02-07

The in situ and direct extraction, desorption and ionization of in-gel intact proteins after electrophoresis has been achieved by carbon nanotubes (CNTs)-modified paper spray mass spectrometry at ambient conditions. Characteristics of CNTs (including larger surface area, smaller pore diameter and enhanced conductivity) were endowed to the porous filter paper substrate by uniformly dispersing the CNTs on the filter paper. Upon applying electric potential to the CNTs-modified paper, the in-gel proteins were extracted from the gel and subsequently migrated to the tip of the filter paper by electrophoresis-like behavior for paper spray ionization, which was monitored by extracted ion chronograms. The characterizations of modified filter papers and CNTs nanoparticles further confirmed the role of CNTs in in-gel protein extraction, protein migration as well as spray ionization at the paper tip. Under optimized conditions, a mixture of cytochrome c, lysozyme and myoglobin was successfully separated by native electrophoresis and subsequently analysed by the present method, showing a limit of detection of 10 ng per gel band. The present strategy offers a new pathway for the direct detection of in-gel intact proteins at ambient conditions without any pre-treatment (e.g. digestion, chemical extraction and desalting), which exhibits potential applications in top-down proteomics.

Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

PubMed Central

Wilkins, Joanna C.; Homer, Karen A.; Beighton, David

2001-01-01

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo. PMID:11472910

Dehumidifier assisted drying of a model fruit pulp-based gel and sensory attributes.

PubMed

Tiwari, Shipra; Ravi, Ramasamy; Bhattacharya, Suvendu

2012-07-01

Model fruit pulp-based gels were prepared by varying mango pulp (0% to 50%), sucrose (0% to 20%), and agar (1% to 3%) and according to a response surface experimental design followed by drying at a low temperature of 40 °C upto 15 h in a tray dryer assisted by a dehumidifier. The moisture content, shrinkage (SHR), and rheological parameters (failure strain, failure stress (FS), firmness, and energy for compression) were determined as a function of drying time. The composition of gel, particularly the agar content had a prominent effect on the characteristics of the dried gel. Detailed descriptive sensory analysis employing principle component analysis (PCA) biplot indicated two distinct groups of attributes; the first group comprised initial and final moisture contents, extent of moisture removal (EMR), and shrinkage. The fracture stress and energy formed the second group. The analysis of variance for failure stress showed that it depended only on the positive linear and quadratic effects of agar (significant at P ≤ 0.01 and 0.05, respectively). The theoretically predicted extent of moisture removal at 95.6% could be achieved when the level of agar was 1.2%; pulp and sucrose levels were also close to their lowest levels of 3.6% and 0.04%, respectively. Scope exists to develop gel-based fruit analogues wherein an appropriate hydrocolloid can be employed along with fruit juice/pulp. To provide a reasonable shelf-life of the developed intermediate moisture containing product, dehumidifier assisted drying is a pragmatic approach that affects sensory and rheological attributes of the dried fruit analogue. © 2012 Institute of Food Technologists®

Factors affecting the separation performance of proteins in capillary electrophoresis.

PubMed

Zhu, Yueping; Li, Zhenqing; Wang, Ping; Shen, Lisong; Zhang, Dawei; Yamaguchi, Yoshinori

2018-04-15

Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min. Copyright © 2018 Elsevier B.V. All rights reserved.

Difference gel electrophoresis (DiGE) identifies differentially expressed proteins in endoscopically-collected pancreatic fluid

PubMed Central

Paulo, Joao A.; Lee, Linda S.; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

2012-01-01

Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis may offer insights into the development and progression of the disease. The endoscopic pancreas function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DiGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe chronic pancreatitis and three chronic abdominal pain controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DiGE and LC-MS/MS, were compared. This DiGE-LC-MS/MS analysis reveals proteins that are differentially expressed in chronic pancreatitis compared to chronic abdominal pain controls. Proteins with higher abundance in pancreatic fluid from chronic pancreatitis individuals include: actin, desmoplankin, alpha-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, alpha-1-antichymotrypsin, alpha-2-macroglobulin, Arp2/3 subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DiGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis, however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy E; Singh, Anup K; Throckmorton, Daniel J

2015-02-24

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

2013-09-03

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Use of agar agar stabilized milled zero-valent iron particles for in situ groundwater remediation

NASA Astrophysics Data System (ADS)

Schmid, Doris; Velimirović, Milica; Wagner, Stephan; Micić Batka, Vesna; von der Kammer, Frank; Hofmann, Thilo

2015-04-01

A major obstacle for use of nanoscale zero-valent iron (nZVI) particles as a nontoxic material for effective in situ degradation of chlorinated aliphatic hydrocarbons (CAHs) is the high production cost. For that reason, submicro-scale milled zero-valent iron particles were recently developed (milled ZVI, UVR-FIA, Germany) by grinding macroscopic raw materials of elementary iron as a cheaper alternative to products produced by solid-state reduction. However, milled ZVI particles tend to aggregate and due to the rather large particle size (d50= 11.9 µm) also rapidly sediment. To prevent aggregation and consequently sedimentation of milled ZVI particles and therefore improve the mobility after in situ application, the use of a stabilizer is considered in literature as a most promising option. In this study, milled ZVI particles (1 g L-1 of particle concentration) were stabilized by environmentally friendly polymer agar agar (>0.5 g L-1), which had a positive impact on the milled ZVI stability. Sedimentation rate was significantly decreased by increasing the suspension viscosity. Column transport experiments were performed for bare and agar agar stabilized milled ZVI particles in commercially available fine grained quartz sand (DORSILIT® Nr.8, Gebrüder Dorfner GmbH Co, Germany) and different porous media collected from brownfields. The experiments were carried out under field relevant injection conditions of 100 m d-1. The maximal travel distance (LT) of less than 10 cm was determined for non-stabilized suspension in fine grained quartz sand, while agar agar (1 g L-1) stabilized milled ZVI suspension revealed LT of 12 m. Similar results were observed for porous media from brownfields showing that mobility of agar agar stabilized particle suspensions was significantly improved compared to bare particles. Based on the mobility data, agar agar stabilized milled zero-valent iron particles could be used for in situ application. Finally, lab-scale batch degradation

Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Oenococcus oeni from different wine-producing regions of China.

PubMed

Wang, Tao; Li, Hua; Wang, Hua; Su, Jing

2015-04-16

The present study established a typing method with NotI-based pulsed-field gel electrophoresis (PFGE) and stress response gene schemed multilocus sequence typing (MLST) for 55 Oenococcus oeni strains isolated from six individual regions in China and two model strains PSU-1 (CP000411) and ATCC BAA-1163 (AAUV00000000). Seven stress response genes, cfa, clpL, clpP, ctsR, mleA, mleP and omrA, were selected for MLST testing, and positive selective pressure was detected for these genes. Furthermore, both methods separated the strains into two clusters. The PFGE clusters are correlated with the region, whereas the sequence types (STs) formed by the MLST confirm the two clusters identified by PFGE. In addition, the population structure was a mixture of evolutionary pathways, and the strains exhibited both clonal and panmictic characteristics. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

PubMed

Anumula, K R; Du, P

1999-11-15

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. Copyright 1999 Academic Press.

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota.

PubMed

Hoyles, Lesley; Clear, Jessica A; McCartney, Anne L

2013-08-01

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intensity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Starbursts and Wispy Drops : Surfactants Spreading on Gel Substrates

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Shomeek; Daniels, Karen; Behringer, Robert

2005-11-01

We report a phase diagram for a novel instability seen in drops of nonionic surfactant solution (Triton X-305) spreading on viscoelastic agar gel substrate . This system allows us to examine the effect of varying the effective fluidity/stiffness of aqueous substrates. The morphology is strongly affected by the substrate fluidity, ranging from spreading starbursts of arms on weak gels, to wispy drops on intermediate strength gels, to circular drops on stiff gels. We analyze the dynamics of spreading in the starburst phase, where the arm length grows as t ^3/4 at early times, independent of the gel strength and surfactant concentration. The number of arms is proportional to the surfactant concentration and inversely proportional to the gel strength. Ongoing work is exploring the effects of changing the drop volume.

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

PubMed Central

Goo, Velma Y. L.; Ching, George Q. L.; Gooch, John M.

1973-01-01

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar. PMID:4584576

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.

PubMed

Jobim, M I M; Trein, C; Zirkler, H; Gregory, R M; Sieme, H; Mattos, R C

2011-09-01

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability. Copyright © 2011 Elsevier Inc. All rights reserved.

Western blotting using capillary electrophoresis.

PubMed

Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

2011-02-15

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

Electron microscopy of antigen precipitates extracted from gel diffusion plates

PubMed Central

Watson, D. H.; Le Bouvier, G. L.; Tomlinson, J. A.; Walkey, D. G. A.

1966-01-01

A method is described whereby material from virus precipitin lines from agar gel diffusion plates may be examined in the electron microscope by a negative staining technique. ImagesFIGS. 1-2FIGS. 3-4 PMID:4286708

Two Distinct Clones of Methicillin-Resistant Staphylococcus aureus (MRSA) with the Same USA300 Pulsed-Field Gel Electrophoresis Profile: a Potential Pitfall for Identification of USA300 Community-Associated MRSA▿

PubMed Central

Larsen, Anders Rhod; Goering, Richard; Stegger, Marc; Lindsay, Jodi A.; Gould, Katherine A.; Hinds, Jason; Sørum, Marit; Westh, Henrik; Boye, Kit; Skov, Robert

2009-01-01

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones. PMID:19759225

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

PubMed

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis.

PubMed

Pang, H M; Yeung, E S

2000-08-01

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50 degrees C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.

Back to the kitchen: food-grade agar is a low-cost alternative to bacteriological agar.

PubMed

Petrovski, Steve; Tillett, Daniel

2012-10-15

Food-grade agar can be used as a low-cost substitute for bacteriological agar in the preparation of solid microbial media. No difference was observed in the colony morphology, growth rate, or viability of bacteria grown on solid media prepared using food-grade agar as compared with using bacteriological-grade agar. This simple tip can reduce the cost of the most common solid media by 80% or more. Copyright © 2012 Elsevier Inc. All rights reserved.

Electrophoresis for the analysis of heparin purity and quality

PubMed Central

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J.

2012-01-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. PMID:22736353

Dynamic computer simulations of electrophoresis: three decades of active research.

PubMed

Thormann, Wolfgang; Caslavska, Jitka; Breadmore, Michael C; Mosher, Richard A

2009-06-01

Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

PubMed Central

Haebel, S.; Jensen, C.; Andersen, S. O.; Roepstorff, P.

1995-01-01

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants. PMID:7795523

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

PubMed

Haebel, S; Jensen, C; Andersen, S O; Roepstorff, P

1995-03-01

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.

Extraction of agar from Gelidium sesquipedale (Rhodopyta) and surface characterization of agar based films.

PubMed

Guerrero, P; Etxabide, A; Leceta, I; Peñalba, M; de la Caba, K

2014-01-01

The chemical structure of the agar obtained from Gelidium sesquipedale (Rhodophyta) has been determined by (13)C nuclear magnetic resonance ((13)C NMR) and Fourier transform infrared spectroscopy (FTIR). Agar (AG) films with different amounts of soy protein isolate (SPI) were prepared using a thermo-moulding method, and transparent and hydrophobic films were obtained and characterized. FTIR analysis provided a detailed description of the binding groups present in the films, such as carboxylic, hydroxyl and sulfonate groups, while the surface composition was examined using X-ray photoelectron spectroscopy (XPS). The changes observed by FTIR and XPS spectra suggested interactions between functional groups of agar and SPI. This is a novel approach to the characterization of agar-based films and provides knowledge about the compatibility of agar and soy protein for further investigation of the functional properties of biodegradable films based on these biopolymers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

PubMed

Gogoi, Purnima; Borah, Probodh; Hussain, Iftikar; Das, Leena; Hazarika, Girin; Tamuly, Shantanu; Barkalita, Luit Moni

2018-05-01

A total of 12 Salmonella isolates belonging to different serovars, viz , Salmonella enterica serovar Enteritidis ( n = 4), Salmonella enterica serovar Weltevreden ( n = 4), Salmonella enterica serovar Newport ( n = 1), Salmonella enterica serovar Litchifield ( n = 1), and untypeable strains ( n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz , repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR. Copyright © 2018 American Society for Microbiology.

Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

PubMed

Herwig, Ela; Marchetti-Deschmann, Martina; Wenz, Christian; Rüfer, Andreas; Redl, Heinz; Bahrami, Soheyl; Allmaier, Günter

2015-06-01

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Multi-chamber electroosmosis using textile reinforced agar membranes--A promising concept for the future of hemodialysis.

PubMed

Kofler, Markus; Lenninger, Margit; Mayer, Gert; Neuwirt, Hannes; Grimm, Michael; Bechtold, Thomas

2016-01-20

Renal replacement therapy options are limited to hemodialysis and peritoneal dialysis (70% of US patients) or renal transplantation. Diffusion processes are the main physico-chemical principle behind hemodialysis. An alternative way to achieve liquid flow through membranes bases on the electroosmotic flow which is observed as electrokinetic phenomenon in porous membranes which bear surface charges. Agar consists of the non-ionic agarose and the negatively charged agaropectine thus an electroosmotic flux is observed in analytical electrophoresis. In this study the potential electroosmosis on textile reinforced agar membranes as separation method was investigated. Using a five-chamber electrolysis cell and an agar membrane/cellulose fabric composite an intensive electroosmotic flow of 1-2 ml cm(2) h(-1) at 100 mA cell current could be observed. The movement of cations in the negatively charged agar structure led to an intensive electroosmotic flux, which also transported uncharged molecules such as urea, glucose through the membrane. Separation of uncharged low molecular weight molecules is determined by the membrane characteristic. The transport of ions (K(+), PO4(3-), creatinine) and uncharged molecules (urea, glucose) in electroosmotic separation experiments was monitored using a pH 5.5 phosphate electrolyte with the aim to assess the overall transport processes in the electrochemical cell. The results demonstrate the potential of the method for filtration of biological fluids in the absence of external pressure or high shear rates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

PubMed Central

Lopez, Isabel; Ruiz-Larrea, Fernanda; Cocolin, Luca; Orr, Erica; Phister, Trevor; Marshall, Megan; VanderGheynst, Jean; Mills, David A.

2003-01-01

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria. PMID:14602643

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

PubMed

Campioni, Fábio; Falcão, Juliana P

2014-06-01

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Simple Protocol for Secondary School Hands-On Activity: Electrophoresis of Pre-Stained Nucleic Acids on Agar-Agar Borate Gels

ERIC Educational Resources Information Center

Britos, Leticia; Goyenola, Guillermo; Orono, Silvia Umpierrez

2004-01-01

An extremely simple, inexpensive, and safe method is presented, which emulates nucleic acids isolation and electrophoretic analysis as performed in a research environment, in the context of a secondary school hands-on activity. The protocol is amenable to an interdisciplinary approach, taking into consideration the electrical and chemical…

Isolation and characterization of a novel agarase-producing Pseudoalteromonas spp. bacterium from the guts of spiny turban shells.

PubMed

Oh, Young Hoon; Jung, Changkyou; Lee, Jinwon

2011-08-01

An agar-degrading bacterium was isolated from the guts of spiny turban shells. It was identified as a Pseudoalteromonas species and named Pseudoalteromonas sp. JYBCL 1. The viscosity of the inoculated agar medium decreased by more than 60% after 20 h cultivation. The agarase produced by the isolate had optimal activities at 35 degrees C and pH 7. The enzyme had extremely strong resistance to ionic stress compared with other known agarases. Its molecular mass was estimated at about 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The agarase could saccharify Gelidium amansii directly, with an efficiency about half that compared with agar saccharification.

Multilocus Sequence Typing Has Better Discriminatory Ability for Typing Vibrio cholerae than Does Pulsed-Field Gel Electrophoresis and Provides a Measure of Phylogenetic Relatedness

PubMed Central

Kotetishvili, Mamuka; Stine, O. Colin; Chen, Yuansha; Kreger, Arnold; Sulakvelidze, Alexander; Sozhamannan, Shanmuga; Morris, Jr., J. Glenn

2003-01-01

Twenty-two Vibrio cholerae isolates, including some from “epidemic” (O1 and O139) and “nonepidemic” serogroups, were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) by using three housekeeping genes, gyrB, pgm, and recA; sequence data were also obtained for the virulence-associated genes tcpA, ctxA, and ctxB. Even with the small number of loci used, MLST had better discriminatory ability than did PFGE. On MLST analysis, there was clear clustering of epidemic serogroups; much greater diversity was seen among tcpA- and ctxAB-positive V. cholerae strains from other, nonepidemic serogroups, with a number of tcpA and ctxAB alleles identified. PMID:12734277

Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yinfa, Ma.

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will bemore » described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.« less

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

PubMed

Jasik, Agnieszka; Reichert, Michal

2006-05-01

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.

Electrophoresis for the analysis of heparin purity and quality.

PubMed

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

2012-06-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

PubMed Central

Nübel, U; Engelen, B; Felske, A; Snaidr, J; Wieshuber, A; Amann, R I; Ludwig, W; Backhaus, H

1996-01-01

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts. PMID:8824607

Microchip capillary gel electrophoresis using programmed field strength gradients for the ultra-fast analysis of genetically modified organisms in soybeans.

PubMed

Kim, Yun-Jeong; Chae, Joon-Seok; Chang, Jun Keun; Kang, Seong Ho

2005-08-12

We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11 s of the microchip. The MCGE-PFSG method was a program that changes the electric field strength during GMO analysis, and was also applied to the ultra-fast analysis of PCR products. Compared to MCGE using a conventional and constantly applied electric field, the MCGE-PFSG analysis generated faster results without the loss of resolving power and reproducibility for specific DNA fragments (100- and 250-bp DNA) of GM-soybeans. The MCGE-PFSG technique may prove to be a new tool in the GMO analysis due to its speed, simplicity, and high efficiency.

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.

A brief review of other notable protein detection methods on acrylamide gels.

PubMed

Kurien, Biji T; Scofield, R Hal

2012-01-01

Several methods have been described to stain proteins analyzed on acrylamide gels. These include ultrasensitive protein detection in one-dimensional and two-dimensional gel electrophoresis using a fluorescent product from the fungus Epicoccum nigrum; a fluorescence-based Coomassie Blue protein staining; visualization of proteins in acrylamide gels using ultraviolet illumination; fluorescence visualization of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; and increasing the sensitivity four- to sixfold for detecting trace proteins in dye or silver stained polyacrylamide gels using polyethylene glycol 6000. All these methods are reviewed briefly in this chapter.

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.

PubMed

Wang, Fangfang; Huang, Lingyun; Na, Na; He, Dacheng; Sun, Dezhi; Ouyang, Jin

2012-05-21

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.

Determination of the molecular weight of human gamma-3 chains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate

PubMed Central

Virella, G.; Parkhouse, R. M. E.

1972-01-01

The molecular weights (mol. wt) for heavy chains of human IgG were estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Polyclonal IgG and monoclonal IgG proteins of different subclasses were extensively reduced with 50 mM dithioerythritol, in the presence of 2 per cent sodium dodecyl sulphate, at 100°. Four control proteins of known mol. wt (cytochrome C, chymotrypsinogen A, egg albumin, and serum albumin) were used to construct a linear plot of electrophoretic mobility versus log mol. wt. From this plot, the following mol. wts were calculated: 53,650±700 for polyclonal IgG; 54,200±1065 for γ1, γ2, and γ4 chains, and 60,950±585 for γ3 chains. Those results confirm the larger size of γ3 chains reported by Saluk and Clem (1971). PMID:4346255

An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling.

PubMed

Hao, Ruijie; Adoligbe, Camus; Jiang, Bijie; Zhao, Xianlin; Gui, Linsheng; Qu, Kaixing; Wu, Sen; Zan, Linsen

2015-01-01

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.

Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.

PubMed

Pan, Tzu-Yu; Chiou, Shyh-Shin; Wang, Chun-Chi; Wu, Shou-Mei

2014-12-01

An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach.

PubMed

Rouquié, David; Capt, Annabelle; Eby, William H; Sekar, Vaithilingam; Hérouet-Guicheney, Corinne

2010-12-01

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context. Copyright © 2010 Elsevier Inc. All rights reserved.

48 CFR 401.371 - AGAR Advisories.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false AGAR Advisories. 401.371 Section 401.371 Federal Acquisition Regulations System DEPARTMENT OF AGRICULTURE GENERAL AGRICULTURE ACQUISITION REGULATION SYSTEM Agency Acquisition Regulations 401.371 AGAR Advisories. The SPE may issue AGAR...

A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins

PubMed Central

Tastet, Christophe; Lescuyer, Pierre; Diemer, Hélène; Luche, Sylvie; van Dorsselaer, Alain; Rabilloud, Thierry

2003-01-01

A new, versatile, multiphasic buffer system for high resolution sodium dodecyl sulfatepolyacrylamide gel electrophoresis of proteins in the relative molecular weight Mw range of 300,000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above Mw range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pKa, as the buffering counter ion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6–200 kDa Mw range, with minimal difficulties in the post electrophoretic identification processes. PMID:12783456

Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikuta, K.; Luftig, R.B.

1988-01-01

The authors detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using /sup 32/P/sub i/-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the /sup 32/P label added to infected cells. When immunoprecipitates from M-MuLV lysates labeled with /sup 32/P/sub i/ were compared with those labeled with (/sup 35/S)methionine, it was calculated that the degree of phosphorylation at themore » p30 domain of Pr65/sup gag/ was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the /sup 32/P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the (/sup 35/S) methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with > 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with (/sup 14/C)sesrine, (/sup 35/S)methionine, and /sup 32/P/sub i/, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH/sub 2/-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.« less

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws.

PubMed

Frazer, G S; Bucci, D M; Brooks, C L

1996-11-01

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved

Screening fungicides for use in fish culture: Evaluation of the agar plug transfer, cellophane transfer, and agar dilution methods

USGS Publications Warehouse

Bailey, Tom A.

1983-01-01

The reliability, reproducibility, and usefulness of three screening methods -- the cellophane transfer, the agar plug transfer, and the agar dilution -- to screen aquatic fungicides were evaluated. Achlya flagellata and Saprolegnia hypogyna were exposed to 1, 10, and 100 mg/L of malachite green to test each method. The cellophane transfer and agar plug transfer techniques had similar reliability and reproducibility in rating fungicidal activity, and were both superior to the agar dilution technique. The agar plug transfer and agar dilution techniques adequately projected in vivo activity of malachite green, but the cellophane transfer technique overestimated its activity. Overall, the agar plug transfer technique most accurately rated the activity of malachite green and was the easiest test to perform. It therefore appears to be the method of choice for testing aquatic fungicides.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support.

PubMed

Rehman, Haneef Ur; Aman, Afsheen; Zohra, Raheela Rahmat; Qader, Shah Ali Ul

2014-02-15

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

PubMed Central

Meroth, Christiane B.; Walter, Jens; Hertel, Christian; Brandt, Markus J.; Hammes, Walter P.

2003-01-01

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. PMID:12514030

Fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturing gradient gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephen, J.R.; Chang, Y.J.; MacNaughton, S.J.

Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. Themore » reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms.« less

Two-Dimensional Differential In-Gel Electrophoresis Proteomic Approaches Reveal Urine Candidate Biomarkers in Pediatric Obstructive Sleep Apnea

PubMed Central

Gozal, David; Jortani, Saeed; Snow, Ayelet B.; Kheirandish-Gozal, Leila; Bhattacharjee, Rakesh; Kim, Jinkwan; Capdevila, Oscar Sans

2009-01-01

Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children. Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA. Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA. Measurements and Main Results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver–operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%. Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted. PMID:19797158

Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

PubMed

Mao, Ping; Hu, Yuanliang; Liao, Tingting; Wang, Zhaoting; Zhao, Shumiao; Liang, Yunxiang; Hu, Yongmei

2017-04-28

The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

PubMed

Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

2012-08-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

PubMed Central

Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, Ângela; Dealtry, Simone; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2012-01-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

Standard Nutrient Agar 1 as a substitute for blood-supplemented Müller-Hinton agar for antibiograms in developing countries.

PubMed

Niederstebruch, N; Sixt, D

2013-02-01

In the industrial world, the agar diffusion test is a standard procedure for the susceptibility testing of bacteria isolates. Beta-hemolytic Streptococcus spp. are tested with Müller-Hinton agar supplemented with 5 % blood, a so-called blood agar. The results are interpreted using standardized tables, which only exist for this type of nutrient matrix. Because of a number difficulties, both with respect to technical issues and to manual skills, blood agar is not a feasible option in many developing countries. Beta-hemolytic Streptococcus spp. also grow on Standard Nutrient Agar 1 (StNA1). This suggests using that type of nutrient medium for running agar diffusion tests. However, there are no standardized tables that can be used for interpreting the diameters of the zones of inhibition on StNA1 1. Using the existing standardized tables for blood agar to interpret cultures on StNA1 1 would be of great benefit under such circumstances where blood agar is not available. With this in mind, we conducted comparative tests to evaluate the growth characteristics of beta-hemolytic Streptococcus spp. on StNA1 1 compared to Müller-Hinton agar supplemented with 5 % sheep blood. In this study, we were able to show that beta-hemolytic Streptococcus spp. develop similar zones of inhibition on blood agar and on StNA1 1. Therefore, it is suggested that, for the interpretation of antibiograms of beta-hemolytic Streptococcus spp. performed on StNA1 1, the standard tables for blood agar can be used.

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

PubMed

Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

2011-07-01

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

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

Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

PubMed

Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

2016-07-01

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Use of agar/glycerol and agar/glycerol/water as a translucent brain simulant for ballistic testing.

PubMed

Falland-Cheung, Lisa; Waddell, J Neil; Lazarjan, Milad Soltanipour; Jermy, Mark C; Winter, Taylor; Tong, Darryl; Brunton, Paul A

2017-01-01

The suitability of agar/glycerol/water and agar/glycerol mixtures as brain simulants was investigated. Test specimens (n=15) (50x27×37mm) were fabricated for these different mixtures and conditioned to 12°C, 22°C, and 26°C prior to testing. For comparison, fresh deer brain specimens (n=20) were sourced and prepared to the same dimensions as the agar/glycerol(/water) mixtures and conditioned to 12°C and 37°C. High impact tests were carried out with a 0.22-caliber air rifle pellet and a high-speed camera was used to record the projectile as it passed through the specimens, allowing for energy loss and vertical displacement velocity calculation. Although the agar/glycerol/water mixture presented with similar vertical expansion and contraction of the specimens to the warm and cold deer brains, a two-fold decrease of the vertical expansion and contraction was noticed with the agar/glycerol specimens. Also considerably less extrusion of this mixture out of the exit and entry sides after specimen penetration was observed. Of the simulants tested, agar/glycerol/water was the most suitable brain simulant for ballistic testing and impact studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polymerase chain reaction-based denaturing gradient gel electrophoresis in the evaluation of oral microbiota.

PubMed

Li, Y; Saxena, D; Barnes, V M; Trivedi, H M; Ge, Y; Xu, T

2006-10-01

Clinical evaluation of oral microbial reduction after a standard prophylactic treatment has traditionally been based on bacterial cultivation methods. However, not all microbes in saliva or dental plaque can be cultivated. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacterial species present in the oral cavity. This study sought to evaluate the oral microbial changes that occurred after a standard prophylactic treatment with a conventional oral care product using PCR-DGGE. Twelve healthy adults participated in the study. Pooled plaque samples were collected at baseline, 24 h after prophylaxis (T1), and 4 days after toothbrushing with fluoride toothpaste (T4). The total microbial genomic DNA of the plaque was isolated. PCR was performed with a set of universal bacterial 16S rDNA primers. The PCR-amplified 16S rDNA fragments were separated by DGGE. The effects of the treatment and of dental brushing were assessed by comparing the PCR-DGGE fingerprinting profiles. The mean numbers of detected PCR amplicons were 22.3 +/- 6.1 for the baseline group, 13.0 +/- 3.1 for the T1 group, and 13.5 +/- 4.3 for the T4 group; the differences among the three groups were statistically significant (P < 0.01). The study also found a significant difference in the mean similarities of microbial profiles between the baseline and the treatment groups (P < 0.001). PCR-based DGGE has been shown to be an excellent means of rapidly and accurately assessing oral microbial changes in this clinical study.

Evaluation and optimisation of preparative semi-automated electrophoresis systems for Illumina library preparation.

PubMed

Quail, Michael A; Gu, Yong; Swerdlow, Harold; Mayho, Matthew

2012-12-01

Size selection can be a critical step in preparation of next-generation sequencing libraries. Traditional methods employing gel electrophoresis lack reproducibility, are labour intensive, do not scale well and employ hazardous interchelating dyes. In a high-throughput setting, solid-phase reversible immobilisation beads are commonly used for size-selection, but result in quite a broad fragment size range. We have evaluated and optimised the use of two semi-automated preparative DNA electrophoresis systems, the Caliper Labchip XT and the Sage Science Pippin Prep, for size selection of Illumina sequencing libraries. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

PubMed

Zsolnai, A; Orbán, L; Chrambach, A

1993-03-01

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

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.

Preparative electrophoresis with on-column optical fiber monitoring and direct elution into a minimized volume.

PubMed

Jackson, George W; Willson, Richard

2005-11-01

A "column-format" preparative electrophoresis device which obviates the need for gel extraction or secondary electro-elution steps is described. Separated biomolecules are continuously detected and eluted directly into a minimal volume of free solution for subsequent use. An optical fiber allows the species of interest to be detected just prior to elution from the gel column, and a small collection volume is created by addition of an ion-exchange membrane near the end of the column.

Identification of inflammation-related proteins in a murine colitis model by 2D fluorescence difference gel electrophoresis and mass spectrometry.

PubMed

Naito, Yuji; Takagi, Tomohisa; Okada, Hitomi; Omatsu, Tatsushi; Mizushima, Katsura; Handa, Osamu; Kokura, Satoshi; Ichikawa, Hiroshi; Fujiwake, Hideshi; Yoshikawa, Toshikazu

2010-05-01

The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis. 2D fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Acute colitis was induced by 8.0% dextran sodium sulfate (DSS) given p.o. for 7 days. Among a total of seven protein spots showing differential expression, we identified five different proteins, of which two were upregulated and three downregulated in colitis in comparison to normal mucosa, using the MASCOT search engine. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase 2 and serpin b1a were upregulated proteins, and protein disulfide-isomerase A3, peroxiredoxin-6 and vimentin were identified as downregulated proteins. These identified proteins may be responsible for the development of the intestinal inflammation. 2D-DIGE and MALDI-TOF mass spectrometry are useful in the search for the differentially expressed proteins.

An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling

PubMed Central

Hao, Ruijie; Adoligbe, Camus; Jiang, Bijie; Zhao, Xianlin; Gui, Linsheng; Qu, Kaixing; Wu, Sen; Zan, Linsen

2015-01-01

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method. PMID:25893432

Comparison of chromogenic Biolog Rainbow agar Shigella/Aeromonas with xylose lysine desoxycholate agar for isolation and detection of Shigella spp. from foods.

PubMed

Zhang, Guodong; Lampel, Keith A

2010-08-01

Shigella outbreaks are widely reported throughout the world. However, it remains a challenge to isolate Shigella spp. from foods by using conventional microbiological media. The main objective of this study was to determine the effectiveness of a novel chromogenic medium, Rainbow agar Shigella/Aeromonas (Rainbow agar), for the isolation and detection of Shigella spp. in foods. All four Shigella species, S. sonnei, S. flexneri, S. dysenteriae, and S. boydii, were studied. Rainbow agar was compared with tryptic soy agar, xylose lysine desoxycholate agar (XLD), and Salmonella Shigella agar (SSA) for enumeration of Shigella spp. in pure culture. This chromogenic agar and XLD were also used to isolate Shigella spp. in artificially contaminated foods (4.8 log CFU/g of food), including lettuce, parsley, cilantro, spinach, potato salad, and shrimp. The inhibitory effect on Shigella growth by Rainbow agar was between that of XLD and SSA. All vegetables studied showed a moderately high background microflora on XLD and Rainbow agar. With artificially inoculated produce, Rainbow agar recovered about 1 to 2 log CFU more S. sonnei, S. dysenteriae, and S. boydii per g of food than did XLD. For potato salad and shrimp, which had low background microflora on Rainbow agar, Rainbow agar was slightly better in recovering Shigella spp. than XLD was in most cases. However, we found that the addition of streptomycin (6.25 mg/liter) to Rainbow agar could facilitate the isolation of Shigella in vegetables tested. In conclusion, Rainbow agar was a much more effective medium than was XLD for the isolation of Shigella spp. from foods.

Separation and characterization of high-density lipoprotein subpopulations by gel permeation chromatography.

PubMed

Clifton, P M; MacKinnon, A M; Barter, P J

1987-02-20

High-density lipoproteins (HDL) contain at least five distinct subpopulations when analyzed by gradient gel electrophoresis. This report represents the first description of a simple technique for isolating these subpopulations of HDL in quantities sufficient to enable characterization in terms of particle size, apolipoprotein AI and apolipoprotein AII content and chemical composition. Lipoproteins were separated and subfractionated on a column of Superose 6B using a fast protein liquid chromatography system. Five normal subjects were studied: HDL2b and HDL3a were isolated as essentially single subpopulations from all subjects, while HDL2a could be isolated from only three of the subjects. HDL3b was isolated in a relatively impure form (70%) from all subjects. Identical subpopulations were identified in each subject by gradient gel electrophoresis of unseparated HDL.

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

PubMed

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut

2013-07-01

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.

Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status

PubMed Central

Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit

2014-01-01

Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study we used a culture-independent method, PCR denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold specific quantitative PCR-analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses, as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p = 0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included S. coelicolor and S. sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included S. hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings. PMID:25331035

Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

PubMed

Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

2014-10-01

The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

Method for preparing hydrous zirconium oxide gels and spherules

DOEpatents

Collins, Jack L.

2003-08-05

Methods for preparing hydrous zirconium oxide spherules, hydrous zirconium oxide gels such as gel slabs, films, capillary and electrophoresis gels, zirconium monohydrogen phosphate spherules, hydrous zirconium oxide spherules having suspendable particles homogeneously embedded within to form a composite sorbent, zirconium monohydrogen phosphate spherules having suspendable particles of at least one different sorbent homogeneously embedded within to form a composite sorbent having a desired crystallinity, zirconium oxide spherules having suspendable particles homogeneously embedded within to form a composite, hydrous zirconium oxide fiber materials, zirconium oxide fiber materials, hydrous zirconium oxide fiber materials having suspendable particles homogeneously embedded within to form a composite, zirconium oxide fiber materials having suspendable particles homogeneously embedded within to form a composite and spherules of barium zirconate. The hydrous zirconium oxide spherules and gel forms prepared by the gel-sphere, internal gelation process are useful as inorganic ion exchangers, catalysts, getters and ceramics.

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

PubMed

Haruta, Shin; Ueno, Shintaro; Egawa, Isao; Hashiguchi, Kazunori; Fujii, Akira; Nagano, Masanobu; Ishii, Masaharu; Igarashi, Yasuo

2006-05-25

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

PubMed

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

tuf-PCR-temporal temperature gradient gel electrophoresis for molecular detection and identification of staphylococci: application to breast milk and neonate gut microbiota.

PubMed

Filleron, Anne; Simon, Margaux; Hantova, Stefaniya; Jacquot, Aurélien; Cambonie, Gilles; Marchandin, Hélène; Jumas-Bilak, Estelle

2014-03-01

Coagulase negative staphylococci (CoNS) are a leading cause of infections in preterm infants, mostly involved in late-onset infection in low birth weight neonates. The epidemiology and pathophysiology of these infections remain unclear, notably because the causing agents are gathered in the artificial CoNS group. The aim of this work was to optimize the study of Staphylococcus species diversity in human breast milk and neonate stool, two sample types with bacterial communities dominated by CoNS, using PCR-temporal temperature gel electrophoresis based on the tuf gene. The optimized protocol identified 18 Staphylococcus species involved in neonate gut microbiota and infections and was applied to cultivation-independent study of breast milk and neonate stool. The efficiency, sensitivity, specificity and species discrimination of the proposed protocol appears suitable for patient follow-up in order to link microbiological data at the community level in milk and stool and interpret them from epidemiological and pathophysiological points of view. Copyright © 2014 Elsevier B.V. All rights reserved.

Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

PubMed

Tanaka, Yasushi; Watanabe, Jun; Mogi, Yoshinobu

2012-08-01

Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

JVirGel 2.0: computational prediction of proteomes separated via two-dimensional gel electrophoresis under consideration of membrane and secreted proteins.

PubMed

Hiller, Karsten; Grote, Andreas; Maneck, Matthias; Münch, Richard; Jahn, Dieter

2006-10-01

After the publication of JVirGel 1.0 in 2003 we got many requests and suggestions from the proteomics community to further improve the performance of the software and to add additional useful new features. The integration of the PrediSi algorithm for the prediction of signal peptides for the Sec-dependent protein export into JVirGel 2.0 allows the exclusion of most exported preproteins from calculated proteomic maps and provides the basis for the calculation of Sec-based secretomes. A tool for the identification of transmembrane helices carrying proteins (JCaMelix) and the prediction of the corresponding membrane proteome was added. Finally, in order to directly compare experimental and calculated proteome data, a function to overlay and evaluate predicted and experimental two-dimensional gels was included. JVirGel 2.0 is freely available as precompiled package for the installation on Windows or Linux operating systems. Furthermore, there is a completely platform-independent Java version available for download. Additionally, we provide a Java Server Pages based version of JVirGel 2.0 which can be operated in nearly all web browsers. All versions are accessible at http://www.jvirgel.de

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

PubMed

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis.

PubMed

Kubota, Kei; Kobayashi, Naoki; Yabuta, Masayuki; Ohara, Motomu; Naito, Toyohiro; Kubo, Takuya; Otsuka, Koji

2017-06-05

This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10kDa marker) of SDS-cGE and increased about 0.5% under a 25°C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase liquid chromatography-mass spectrometry (RPLC-MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5°C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS-cGE, providing information critical to assuring the quality and stability of the biotherapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science.

PubMed

Han, Jun P; Sun, Jing; Wang, Le; Liu, Peng; Zhuang, Bin; Zhao, Lei; Liu, Yao; Li, Cai X

2017-11-01

Microfluidic chips offer significant speed, cost, and sensitivity advantages, but numerous parameters must be optimized to provide microchip electrophoresis detection. Experiments were conducted to study the factors, including sieving matrices (the concentration and type), surface modification, analysis temperature, and electric field strengths, which all impact the effectiveness of microchip electrophoresis detection of DNA samples. Our results showed that the best resolution for ssDNA was observed using 4.5% w/v (7 M urea) lab-fabricated LPA gel, dynamic wall coating of the microchannel, electrophoresis temperatures between 55 and 60°C, and electrical fields between 350 and 450 V/cm on the microchip-based capillary electrophoresis (μCE) system. One base-pair resolution could be achieved in the 19-cm-length microchannel. Furthermore, both 9947A standard genomic DNA and DNA extracted from blood spots were demonstrated to be successfully separated with well-resolved DNA peaks in 8 min. Therefore, the microchip electrophoresis system demonstrated good potential for rapid forensic DNA analysis. © 2017 American Academy of Forensic Sciences.

In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

PubMed

Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

2006-01-01

Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

PubMed

Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

2014-08-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....4600 Ouchterlony agar plate. (a) Identification. An ouchterlony agar plate for clinical use is a device...

21 CFR 866.4600 - Ouchterlony agar plate.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Ouchterlony agar plate. 866.4600 Section 866.4600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED....4600 Ouchterlony agar plate. (a) Identification. An ouchterlony agar plate for clinical use is a device...

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

PubMed

Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen

2013-08-01

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The classes alpha-, beta-, and gamma-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity (average H' = 2.65) when compared to non-impacted sites (average H' = 3.05). There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC), total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact.

Antimicrobial activity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin film.

PubMed

Ghosh, S; Kaushik, R; Nagalakshmi, K; Hoti, S L; Menezes, G A; Harish, B N; Vasan, H N

2010-10-13

Highly stable silver nanoparticles (Ag NPs) in agar-agar (Ag/agar) as inorganic-organic hybrid were obtained as free-standing film by in situ reduction of silver nitrate by ethanol. The antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) was evaluated in a nutrient broth and also in saline solution. In particular, films were repeatedly tested for antimicrobial activity after recycling. UV-vis absorption and TEM studies were carried out on films at different stages and morphological studies on microbes were carried out by SEM. Results showed spherical Ag NPs of size 15-25 nm, having sharp surface plasmon resonance (SPR) band. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans>E. coli>S. aureus, and antimicrobial activity against C. albicans was almost maintained even after the third cycle. Whereas, in case of E. coli and S. aureus there was a sharp decline in antimicrobial activity after the second cycle. Agglomeration of Ag NPs in Ag/agar film on exposure to microbes was observed by TEM studies. Cytotoxic experiments carried out on HeLa cells showed a threshold Ag NPs concentration of 60 μg/mL, much higher than the minimum inhibition concentration of Ag NPs (25.8 μg/mL) for E. coli. The mechanical strength of the film determined by nanoindentation technique showed almost retention of the strength even after repeated cycle. Copyright © 2010 Elsevier Ltd. All rights reserved.

Novel royal jelly proteins identified by gel-based and gel-free proteomics.

PubMed

Han, Bin; Li, Chenxi; Zhang, Lan; Fang, Yu; Feng, Mao; Li, Jianke

2011-09-28

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.

Understanding Electrophoresis through the Investigation of Size, Shape, and Charge of pH Indicators

ERIC Educational Resources Information Center

Brenner, Ryan K.; Hess, Kenneth R.; Morford, Jennifer L.

2015-01-01

A laboratory experiment was designed for upper-level students in a Chemical Analysis course to illustrate the theoretical and practical applications of 0.8% agarose gel electrophoresis and to reinforce an understanding of weak acids/bases using easy-to-visualize pH indicators. The careful choice of indicators included acid and base types with…

Sources of Listeria monocytogenes Contamination in a Cold-Smoked Rainbow Trout Processing Plant Detected by Pulsed-Field Gel Electrophoresis Typing

PubMed Central

Autio, Tiina; Hielm, Sebastian; Miettinen, Maria; Sjöberg, Anna-Maija; Aarnisalo, Kaarina; Björkroth, Johanna; Mattila-Sandholm, Tiina; Korkeala, Hannu

1999-01-01

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating L. monocytogenes. None of the control samples taken in the 5 months after the eradication program was implemented contained L. monocytogenes. PMID:9872773

A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

PubMed

Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

2012-01-01

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

Characterization of agar/soy protein biocomposite films: Effect of agar on the extruded pellets and compression moulded films.

PubMed

Garrido, T; Etxabide, A; Guerrero, P; de la Caba, K

2016-10-20

Agar/soy protein biocomposite films were successfully processed by extrusion and compression moulding, obtaining transparent and homogeneous films. The conformational changes occurred during the extrusion process and the effect of agar on the final properties were analyzed. As shown by differential scanning calorimetry (DSC) and specific mechanical energy (SME) values, during the extrusion process protein denatured and unfolded protein chains could interact with agar. These interactions were analyzed by Fourier transform infrared spectroscopy (FTIR) and the secondary structure was determined from the amide I band. Those interactions were supported by the decrease of film solubility. Furthermore, the good compatibility between agar and soy protein was confirmed by the images from scanning electron microscopy (SEM). Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Multi-gel casting apparatus for vertical polyacrylamide gels with in-built solution flow system and liquid level detectors.

PubMed

Maurye, Praveen; Basu, Arpita; Bandyopadhyay, Tapas Kumar; Biswas, Jayanta Kumar; Mohanty, Bimal Prasana

2017-08-01

PAGE is the most widely used technique for the separation and biochemical analysis of biomolecules. The ever growing field of proteomics and genomics necessitates the analysis of many proteins and nucleic acid samples to understand further about the structure and function of cells. Simultaneous analysis of multiple protein samples often requires casting of many PAGE gels. Several variants of multi-gel casting/electrophoresis apparatuses are frequently used in research laboratories. Requirement of supplementary gels to match the growing demand for analyzing additional protein samples sometimes become a cause of concern. Available apparatuses are not amenable to and therefore, not recommended for any modification to accommodate additional gel casting units other than what is prescribed by the manufacturer. A novel apparatus is described here for casting multiple PAGE gels comprising four detachable components that provide enhanced practicability and performance of the apparatus. This newly modified apparatus promises to be a reliable source for making multiple gels in less time without hassle. Synchronized functioning of unique components broaden the possibilities of developing inexpensive, safe, and time-saving multi-gel casting apparatus. This apparatus can be easily fabricated and modified to accommodate desired number of gel casting units. The estimated cost (∼$300) for fabrication of the main apparatus is very competitive and effortless assembly procedure can be completed within ∼30 min. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

PubMed

Sakurai, R; Sasaki, N; Takano, H; Abe, T; Kawano, S

2000-04-28

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil.

PubMed

Jousset, Alexandre; Lara, Enrique; Nikolausz, Marcell; Harms, Hauke; Chatzinotas, Antonis

2010-02-01

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats. Copyright 2009 Elsevier B.V. All rights reserved.

Comparison of pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae.

PubMed

Ture, M; Altinok, I; Capkin, E

2015-01-01

Biochemical test, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC-PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216-6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC-PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC-PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods. © 2014 John Wiley & Sons Ltd.

Molecular subtyping of Clostridium perfringens by pulsed-field gel electrophoresis to facilitate food-borne-disease outbreak investigations.

PubMed

Maslanka, S E; Kerr, J G; Williams, G; Barbaree, J M; Carson, L A; Miller, J M; Swaminathan, B

1999-07-01

Clostridium perfringens is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported. Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals. Serotyping has been used in the past to assist epidemiologic investigations of C. perfringens outbreaks. However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents. We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of C. perfringens isolates to aid in epidemiologic investigations of food-borne outbreaks. Six restriction endonucleases (SmaI, ApaI, FspI, MluI, KspI, and XbaI) were evaluated with a select panel of C. perfringens strains. SmaI was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to approximately 1,100 kb which provided good discrimination between isolates. Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains. In general, multiple isolates from a single individual had indistinguishable PFGE patterns. Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns. PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by C. perfringens.

[Genotypic variability and persistence of Legionella pulsed-field gel electrophoresis patterns in 16 cooling towers in Shanghai, China].

PubMed

Chen, Ming-liang; Wang, Gang-yi; Chen, Min; Zhou, Hai-jian; Shao, Zhu-jun; Zhang, Xi; Wu, Fan

2010-07-01

To investigate the genotypic characteristics and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns in 16 air-conditioner cooling towers in six different public sites of Shanghai. From May to October, continuous sampling was operated once per month in 2007. Legionella strains isolated from the 16 cooling towers were confirmed by serological and latex agglutination. PFGE was applied for the fingerprinting of the isolates, while the cluster results of PFGE were analyzed by BioNumerics software. 131 strains of Legionella were isolated, including L. pneumophila, L. bozemanae, L. micdadei and L. anisa. 52 distinguishable PFGE patterns were differentiated among the 16 cooling towers, with 37 patterns were owned by just one cooling tower, which was not shared with other cooling towers, while 15 patterns were shared by more than 2 cooling towers. All the cooling towers had ≥ 2 PFGE patterns, while in 13 cooling towers the same PFGE patterns were recovered during the six months. From June to October of 2007, 18 strains of Legionella belonging to the PFGE pattern of LPAs.SH0078 were isolated continuously from 6 cooling towers. This study demonstrated great genotypic diversity and complexity of Legionella in cooling towers. Persistence of the PFGE patterns was observed in 81.25% of the cooling towers. The PFGE pattern of LPAs. SH0078 was distributed widely, suggesting it might be the dominate strain in Shanghai.

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.

PubMed

Piwat, S; Teanpaisan, R

2013-01-01

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances.

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances

PubMed Central

Piwat, S.; Teanpaisan, R.

2013-01-01

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

Investigations on gel forming media use in low gravity bioseparations research

NASA Technical Reports Server (NTRS)

Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine

1989-01-01

Research on gelling media and conditions suitable for the preservation of the spatial configuration of cell suspensions and macromolecular solutions after separation in free fluid during low gravity experiments is presented. The examples studied included free electrophoresis of cells in a cylindrical column and two-phase aqueous polymer separation. Microgravity electrophoresis experiments were simulated by separating model cell types (animal or human) in a vertical density gradient containing low-conductivity buffer, 1.7-6.5 percent Ficoll, 6.8-5.0 percent sucrose, and 1 percent SeaPrep low-melting temperature agarose. Upon cooling, a gel formed in the column and cells could be captured at the forming locations. Two-phase extraction experiments were simulated using 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 percent), maltodextrin (5-7 percent), and gelatin (5-20 percent).

A novel polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages.

PubMed

Cocolin, L; Manzano, M; Aggio, D; Cantoni, C; Comi, G

2001-05-01

A new molecular method consisting of polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis (DGGE) of a small fragment from the 16S rRNA gene identified the Micrococcaceae strains isolated from natural fermented Italian sausages. Lactic acid bacteria, total aerobic mesophilic flora, Enterobacteriaceae and faecal enterococci were also monitored. Micrococcaceaea control strains from international collections were used to optimise the method and 90 strains, isolated from fermented sausages, were identified by biochemical tests and PCR-DGGE. No differences were observed between the methods used. The results reported in this paper prove that Staphylococcus xylosus is the main bacterium involved in fermented sausage production, representing, from the tenth day of ripening, the only Micrococcaceaea species isolated.

Establishment of a Universal Size Standard Strain for Use with the PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocols: Converting the National Databases to the New Size Standard

PubMed Central

Hunter, Susan B.; Vauterin, Paul; Lambert-Fair, Mary Ann; Van Duyne, M. Susan; Kubota, Kristy; Graves, Lewis; Wrigley, Donna; Barrett, Timothy; Ribot, Efrain

2005-01-01

The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described. PMID:15750058

Canine serum protein patterns using high-resolution electrophoresis (HRE).

PubMed

Abate, O; Zanatta, R; Malisano, T; Dotta, U

2000-03-01

Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively. In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information. Copyright 2000 Harcourt Publishers Ltd.

Ultraviolet-B radiation induced crosslinking improves physical properties of cold- and warm-water fish gelatin gels and films

USDA-ARS?s Scientific Manuscript database

Cold- and warm-water fish gelatin granules were exposed to ultraviolet-B radiation for doses up to 29.7 J/cm2. Solutions and films were prepared from the granules. Gel electrophoresis and refractive index were used to examine changes in molecular weight of the samples. Also, the gel strength and rhe...

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

PubMed Central

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

Agar dilution and agar screen with cefoxitin and oxacillin: what is known and what is unknown in detection of meticillin-resistant Staphylococcus aureus.

PubMed

Perez, Leandro Reus Rodrigues; Dias, Cícero; d'Azevedo, Pedro Alves

2008-08-01

In this study we evaluated the performance of the oxacillin agar screen test, and agar dilution tests using cefoxitin and oxacillin antimicrobials, to detect meticillin resistance in Staphylococcus aureus isolates. The presence of the mecA gene, detected by PCR, was used as the standard to which agar screen and agar dilution tests were compared. The best performance was obtained using the agar dilution test (99.4 % accuracy) with breakpoints of 4 mug ml(-1) for oxacillin and 8 mug ml(-1) for cefoxitin, and using the oxacillin agar screen test. Also, a strong correlation between MIC values of cefoxitin and oxacillin permits the use of either drug for detection of meticillin resistance.

Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

PubMed

Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

2005-10-06

We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

An analysis of variation in the long-range genomic organization of the human major histocompatibility complex class II region by pulsed-field gel electrophoresis.

PubMed

Dunham, I; Sargent, C A; Dawkins, R L; Campbell, R D

1989-11-01

The class II region of the human major histocompatibility complex in seven common HLA haplotypes has been analyzed using pulsed-field gel electrophoresis, restriction enzymes that cut genomic DNA infrequently, and Southern blotting. This analysis has revealed that there are differences in the amount of DNA present in the DQ and DR subregions dependent on the haplotype. The class II region of the DR3 haplotype spans approximately 750 kb and has the same amount of DNA as the class II region of the DR5 and DR6 haplotypes. However, the DR2 haplotype has approximately 30 kb more DNA within the DR subregion. The DR4 haplotype has an additional approximately 110 kb of DNA within the DQ or DR subregions compared to the DR3, DR5, and DR6 haplotypes. These haplotype-specific differences could have some bearing both on the analysis of disease susceptibility and on the ability of chromosomes possessing different HLA haplotypes to recombine within the DQ/DR subregions.

New cellular automaton designed to simulate geometration in gel electrophoresis

NASA Astrophysics Data System (ADS)

Krawczyk, M. J.; Kułakowski, K.; Maksymowicz, A. Z.

2002-08-01

We propose a new kind of cellular automaton to simulate transportation of molecules of DNA through agarose gel. Two processes are taken into account: reptation at strong electric field E, described in the particle model, and geometration, i.e. subsequent hookings and releases of long molecules at and from gel fibres. The automaton rules are deterministic and they are designed to describe both processes within one unified approach. Thermal fluctuations are not taken into account. The number of simultaneous hookings is limited by the molecule length. The features of the automaton are: (i) the size of the cell neighbourhood for the automaton rule varies dynamically, from nearest neighbors to the entire molecule; (ii) the length of the time step is determined at each step according to dynamic rules. Calculations are made up to N=244 reptons in a molecule. Two subsequent stages of the motion are found. Firstly, an initial set of random configurations of molecules is transformed into a more ordered phase, where most molecules are elongated along the applied field direction. After some transient time, the mobility μ reaches a constant value. Then, it varies with N as 1/ N for long molecules. The band dispersion varies with time t approximately as Nt1/2. Our results indicate that the well-known plateau of the mobility μ vs. N does not hold at large electric fields.

Rapid in vitro labeling procedures for two-dimensional gel fingerprinting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Y.F.; Fowlks, E.R.

1982-01-15

Improvements of existing in vitro procedures for labeling RNA radioactively, and modifications of the two-dimensional polyacrylamide gel electrophoresis system for making RNA fingerprints are described. These improvements are (a) inactivation of phosphatase with nitric acid at pH 2.0 eliminated the phenol-cholorform extraction step during 5'-end labeling with polynucleotide kinase and (..gamma..-/sup 32/P)ATP; (b) ZnSO/sub 4/ inactivation of R Nase T/sub 1/ results in a highly efficient procedure for 3'-end labeling with T4 ligase and (5'-/sup 32/P)pCp; and (c) a rapid 4-min procedure for variable quantity range of /sup 125/I and RNA results in a qualitative and quantitative sample for high-molecularmore » weight RNA fingerprinting. Thus, these in vitro procedures become rapid and reproducible when combined with two-dimensional gel electrophoresis which eliminates simultaneously labeled impurities. Each labeling procedure is compared, using tobacco mosaic virus, Brome mosaic virus, and polio RNA. A series of Ap-rich oligonucleotides was discovered in the inner genome of Brome mosaic Virus RNA-3.« less

Method for preparing hydrous iron oxide gels and spherules

DOEpatents

Collins, Jack L.; Lauf, Robert J.; Anderson, Kimberly K.

2003-07-29

The present invention is directed to methods for preparing hydrous iron oxide spherules, hydrous iron oxide gels such as gel slabs, films, capillary and electrophoresis gels, iron monohydrogen phosphate spherules, hydrous iron oxide spherules having suspendable particles homogeneously embedded within to form composite sorbents and catalysts, iron monohydrogen phosphate spherules having suspendable particles of at least one different sorbent homogeneously embedded within to form a composite sorbent, iron oxide spherules having suspendable particles homogeneously embedded within to form a composite of hydrous iron oxide fiber materials, iron oxide fiber materials, hydrous iron oxide fiber materials having suspendable particles homogeneously embedded within to form a composite, iron oxide fiber materials having suspendable particles homogeneously embedded within to form a composite, dielectric spherules of barium, strontium, and lead ferrites and mixtures thereof, and composite catalytic spherules of barium or strontium ferrite embedded with oxides of Mg, Zn, Pb, Ce and mixtures thereof. These variations of hydrous iron oxide spherules and gel forms prepared by the gel-sphere, internal gelation process offer more useful forms of inorganic ion exchangers, catalysts, getters, dielectrics, and ceramics.

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

PubMed Central

Kang, D. H.; Siragusa, G. R.

1999-01-01

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media. PMID:10583985

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

PubMed

Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-11-01

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis.

PubMed

Salamatmanesh, Mina; McCudden, Christopher R; McCurdy, Arleigh; Booth, Ronald A

2018-01-01

The International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧25% with an absolute change of no <5g/L for either minimal response or progression. Limited evidence is available to accurately determine the magnitude of change in a monoclonal protein to reflect a true change in clinical status. Here we determined the analytical and biological variability of monoclonal proteins in stable monoclonal gammopathy of undetermined significance (MGUS) patients. Analytical variability (CVa) of normal protein fractions and monoclonal proteins were assessed agarose gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV). Analytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6g/L) and high (32.2g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi. Serial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization.

PubMed

Jenkins, Frank J; Kerr, Charles M; Fouquerel, Elise; Bovbjerg, Dana H; Opresko, Patricia L

2017-07-10

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern 1 , 2 . Here, we present a detailed description of this procedure, with some modifications.

Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis.

PubMed

Hromadnikova, I; Zejskova, L; Doucha, J; Codl, D

2006-11-01

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.

Variable number of tandem repeats and pulsed-field gel electrophoresis cluster analysis of enterohemorrhagic Escherichia coli serovar O157 strains.

PubMed

Yokoyama, Eiji; Uchimura, Masako

2007-11-01

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157.

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis.

PubMed

Podszun, Susann; Vulto, Paul; Heinz, Helene; Hakenberg, Sydney; Hermann, Carsten; Hankemeier, Thomas; Urban, Gerald A

2012-02-07

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.

A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae

PubMed Central

Cooke, Venitia M.; Miles, R. J.; Price, R. G.; Richardson, A. C.

1999-01-01

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter−1. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997

Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

PubMed

Park, Chul; Helm, Richard F; Novak, John T

2008-12-01

The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study.

[Preparing oral dosage form of ketamine in the hospital for simplicity and patient compliance--preparations using agar].

PubMed

Kaneuchi, Miki; Kohri, Naonori; Senbongi, Kaname; Sakai, Hideo; Iseki, Ken

2005-02-01

Ketamine has been widely used in the operation as intravenous and intramuscular injections, since ketamine has dissociative anesthetic properties. When it is given in sub-anesthetic dose, ketamine is known to have an analgesic effect. The analgesic effect is observed for patients with neuropathic pain when administrated not only by injection but also orally. In Japan, since ketamine is not commercially available except injection forms, patients have to take it as solution of injections for the oral medication. Since the solution of injections has extremely bitter taste, patients intensely desire the development of preparations without the bitterness. In the present study, we prepared oral gel dosage forms of ketamine using agar. It is simple to prepare this dosage form, and most pharmacists can prepare it easily in many hospitals. This gel dosage form met content uniformity requirements and the shape of that was maintained intact during the dissolution test (for 10 hours). The release rate was reduced by additions of additives such as sugar and a flavor in the gel. The reason for the reduction in release could be the suppression of ketamine diffusion depended on the micro-viscosity of solution in the gel. The ketamine contents and the release profile of the gel preparations were unchanged at the room temperature for 12-week storage. The gel preparations in this study would be useful for the oral medication of ketamine, since it is easy for patients to carry them when they go out and the intensely bitter taste could be improved by the addition of a flavor.

Separation of Native Allophycocyanin and R-Phycocyanin from Marine Red Macroalga Polysiphonia urceolata by the Polyacrylamide Gel Electrophoresis Performed in Novel Buffer Systems

PubMed Central

Wang, Yu; Gong, Xueqin; Wang, Shumei; Chen, Lixue; Sun, Li

2014-01-01

Three buffer systems of Imidazole−Acetic acid, HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES were designed based on the principle of discontinuous polyacrylamide gel electrophoresis (PAGE) for the native PAGE which could be performed in pH 7.0 and 6.5 in order to analyze and prepare the minor components of allophycocyanin (AP) and R-phycocyanin (R-PC) from marine red macroalga Polysiphonia urceolata. These AP and R-PC phycobiliproteins are easily denatured in alkaline environments. The obtained results demonstrated that the PAGE modes performed in the buffer systems of HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES gave the satisfactory resolution and separation of AP and R-PC proteins. The absorption and fluorescence spectra of the AP and R-PC proteins which were prepared by the established PAGE modes proved that they maintained natural spectroscopic characteristics. The established PAGE modes may also provide useful references and selections for some other proteins that are sensitive to alkaline environments or are not effectively separated by the classical PAGE modes performed normally in alkaline buffer systems. PMID:25166028

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

Borelli's lactritmel agar induces conidiation in rare-macroconidia producing dermatophytic fungi.

PubMed

Ilkit, Macit; Gümral, Ramazan; Döğen, Aylin

2012-10-01

Macroconidia are among the most important indicators used to identify dermatophytic fungi, but several do not usually sporulate and/or produce macroconidia on Sabouraud glucose agar. Specifically, Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely form macroconidia and, therefore, cannot be easily identified. In this study, we investigated the production of macroconidia on nine common laboratory media, including Borelli's lactritmel agar (BLA), modified Borelli's lactritmel agar (MBLA), brain heart infusion agar (BHIA), Christensen's urease agar in Petri dishes (UPA), cornmeal dextrose agar (CMDA), Lowenstein-Jensen agar (LJA), malt extract agar (MEA), oatmeal agar (OA), and potato dextrose agar (PDA). The performance of these media was evaluated using 18 rare-macroconidia producing isolates, including representative of the six species mentioned above. All cultures in this study were incubated at 26°C on the bench, and conidia formation on each was investigated at 5, 10, 15, 20, 25, and 30 days of incubation. BLA apparently improved macroconidia production after 15 days and was the most useful nutrient agar medium to induce these phenotypic characters in daily practice, closely followed by OA, PDA, and MBLA.

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.…

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. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Pulsed-Field Gel Electrophoresis Analysis of Bordetella pertussis Isolates Circulating in Europe from 1998 to 2009

PubMed Central

Advani, Abdolreza; Hallander, Hans O.; Dalby, Tine; Krogfelt, Karen Angeliki; Guiso, Nicole; Njamkepo, Elisabeth; von Könnig, Carl Heinz Wirsing; Riffelmann, Marion; Mooi, Frits R.; Sandven, Per; Lutyńska, Anna; Fry, Norman K.; Mertsola, Jussi

2013-01-01

Between 1998 and 2009, Bordetella pertussis clinical isolates were collected during three periods, i.e., 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), and 2007 to 2009 (n = 140), from nine countries with distinct vaccination programs, i.e., Denmark, Finland, France, Germany, The Netherlands, Norway, Poland, Sweden, and the United Kingdom. Pulsed-field gel electrophoresis (PFGE) analysis was performed according to standardized recommendations for epidemiological typing of B. pertussis. There were 81 different PFGE profiles, five of which (BpSR3, BpSR5, BpSR10, BpSR11, and BpSR12) were observed in 61% of the 396 isolates and shown to be predominant in almost all countries. The major profile, BpSR11, showed a decreasing trend from 25% to 30% in 1998 to 2005 to 13% in 2007 to 2009, and there were increases in BpSR3 and BpSR10 from 0% and 8% to 21% and 22%, respectively. One difference between these profiles is that BpSR11 contains isolates harboring the fim3-2 allele and BpSR3 and BpSR10 contain isolates harboring the fim3-1 allele. The total proportion of the five predominant profiles increased from 44% in 1998 to 2001 to 63% in 2004 to 2005 to 70% in 2007 to 2009. In conclusion, common PFGE profiles were identified in B. pertussis populations circulating in European countries with different vaccination programs and different vaccine coverages. These prevalent isolates contain the novel pertussis toxin promoter ptxP3 allele. However, there is evidence for diversifying selection between ptxP3 strains characterized by distinct PFGE profiles. This work shows that, even within a relatively short time span of 10 years, successful isolates which spread through Europe and cause large shifts in B. pertussis populations may emerge. PMID:23175253

Application of pulsed field gel electrophoresis to the 1993 epidemic of whooping cough in the UK.

PubMed Central

Syedabubakar, S. N.; Matthews, R. C.; Preston, N. W.; Owen, D.; Hillier, V.

1995-01-01

The purpose of this study was to DNA fingerprint the majority (64%) of isolates received at the Pertussis Reference Laboratory during the 1993 whooping cough epidemic by pulsed field gel electrophoresis of Xba I-generated restriction digests. Two DNA restriction patterns, types 1 and 3, predominated (40% and 23%, respectively, of 180 isolates) but type 2, identified in a previous study was notably absent. Twenty-one new DNA types occurred (24% of isolates), some being atypical as bands 155-230 kb were no longer conserved, but there was no statistically significant difference in their incidence in the upswing (June-September) compared to the downswing (October-December) phase of the epidemic. There was a relatively high proportion of new types, compared to type 1, at the peak (September). About 50% of isolates received were from the North Western Region, where 44% of isolates were DNA type 1. Whereas only 1 out of 10 isolates from Scotland were of this type, suggesting some geographic variation. Statistically significant findings included a higher proportion of isolates from female patients (P < 0.01), most marked in the 12-24 months age group (P < 0.05); a higher proportion of infants under 12 months requiring hospital admission compared to older children (P < 0.05); and a greater number of isolates from unvaccinated children (P < 0.01). Analysis of serotype according to four age groups (under 3 months, 3-12 months, 12-24 months and above 2 years) showed statistically significant differences (P < 0.05) with a noticeably lower proportion (38%) of serotype 1,3 in 3-12 months age group and higher prevalence (74%) of serotype 1,3 in the 12-24 months age group. There was no correlation between DNA type and serotype. Images Fig. 2 PMID:7641824

Method for preparing hydrous titanium oxide spherules and other gel forms thereof

DOEpatents

Collins, J.L.

1998-10-13

The present invention are methods for preparing hydrous titanium oxide spherules, hydrous titanium oxide gels such as gel slabs, films, capillary and electrophoresis gels, titanium monohydrogen phosphate spherules, hydrous titanium oxide spherules having suspendible particles homogeneously embedded within to form a composite sorbent, titanium monohydrogen phosphate spherules having suspendible particles of at least one different sorbent homogeneously embedded within to form a composite sorbent having a desired crystallinity, titanium oxide spherules in the form of anatase, brookite or rutile, titanium oxide spherules having suspendible particles homogeneously embedded within to form a composite, hydrous titanium oxide fiber materials, titanium oxide fiber materials, hydrous titanium oxide fiber materials having suspendible particles homogeneously embedded within to form a composite, titanium oxide fiber materials having suspendible particles homogeneously embedded within to form a composite and spherules of barium titanate. These variations of hydrous titanium oxide spherules and gel forms prepared by the gel-sphere, internal gelation process offer more useful forms of inorganic ion exchangers, catalysts, getters and ceramics. 6 figs.

Method for preparing hydrous titanium oxide spherules and other gel forms thereof

DOEpatents

Collins, Jack L.

1998-01-01

The present invention are methods for preparing hydrous titanium oxide spherules, hydrous titanium oxide gels such as gel slabs, films, capillary and electrophoresis gels, titanium monohydrogen phosphate spherules, hydrous titanium oxide spherules having suspendible particles homogeneously embedded within to form a composite sorbent, titanium monohydrogen phosphate spherules having suspendible particles of at least one different sorbent homogeneously embedded within to form a composite sorbent having a desired crystallinity, titanium oxide spherules in the form of anatase, brookite or rutile, titanium oxide spherules having suspendible particles homogeneously embedded within to form a composite, hydrous titanium oxide fiber materials, titanium oxide fiber materials, hydrous titanium oxide fiber materials having suspendible particles homogeneously embedded within to form a composite, titanium oxide fiber materials having suspendible particles homogeneously embedded within to form a composite and spherules of barium titanate. These variations of hydrous titanium oxide spherules and gel forms prepared by the gel-sphere, internal gelation process offer more useful forms of inorganic ion exchangers, catalysts, getters and ceramics.

Green synthesis of gold nanoparticles of different sizes and shapes using agar-agar water solution and femtosecond pulse laser irradiation

NASA Astrophysics Data System (ADS)

Almeida de Matos, Ricardo; da Silva Cordeiro, Thiago; Elgul Samad, Ricardo; Dias Vieira, Nilson; Coronato Courrol, Lilia

2012-11-01

We report a method to create gold nanoparticles of different sizes and shapes using agar-agar water solution and irradiation with light from a xenon lamp, followed by ultrashort laser pulses. No additives, such as solvents, surfactants or reducing agents, were used in the procedure. Laser irradiation (laser ablation) was important to the reduction of the nanoparticles diameter and formation of another shapes. Distilled water was used as solvent and agar-agar (hydrophilic colloid extracted from certain seaweeds) was important for the stabilization of gold nanoparticles, avoiding their agglomeration. The formation of gold nanoparticles was confirmed with ultraviolet-visible absorption and TEM microscopy. The gold nanoparticles acquired spherical, prism, and rod shapes depending on the laser parameters. Variation of laser irradiation parameters as pulse energy, irradiation time and repetition rate was assessed. The relevant mechanisms contributing for the gold nanoparticles production are discussed.

Protein electrophoresis in cranes with presumed insect bite.

PubMed

Hartup, Barry K; Schroeder, Carrie A

2006-06-01

Serum protein electrophoresis (SPE) has emerged as a potentially valuable diagnostic tool in avian medicine; yet, there is limited information regarding SPE in cranes. Since 2000, 20 cases of unilateral periocular or facial soft tissue swelling, blepharitis, feather loss, and ocular or nasal discharge attributed to insect bite hypersensitivity were observed in cranes from a captive breeding center. SPE may be useful for evaluating these lesions. The aim of this study was to characterize the inflammatory response in cranes with hypersensitivity reactions using SPE. Serum samples from 7 cranes diagnosed with hypersensitivity reactions were submitted to a diagnostic laboratory for agarose gel electrophoresis. Results were compared to those in control serum samples obtained from the same cranes during routine physical examination, when they were clinically healthy. Total protein and a- and g-globulin concentrations were significantly increased and albumin/globulin ratios were significantly decreased in serum samples from cranes with hypersensitivity lesions compared with control samples. Using SPE, we documented changes in protein fraction concentrations in cranes with clinical signs of hypersensitivity. The increase in alpha- and gamma-globulin concentrations suggested inflammation and antigenic stimulation, consistent with a Type I hypersensitivity reaction.

Bacterial community structure analysis of sediment in the Sagami River, Japan using a rapid approach based on two-dimensional DNA gel electrophoresis mapping with selective primer pairs.

PubMed

Liu, Guo-hua; Rajendran, Narasimmalu; Amemiya, Takashi; Itoh, Kiminori

2011-11-01

A rapid approach based on two-dimensional DNA gel electrophroesis (2-DGE) mapping with selective primer pairs was employed to analyze bacterial community structure in sediments from upstream, midstream and downstream of Sagami River in Japan. The 2-DGE maps indicated that Alpha- and Delta-proteobacteria were major bacterial populations in the upstream and midstream sediments. Further bacterial community structure analysis showed that richness proportion of Alpha- and Delta-proteobacterial groups reflected a trend toward decreasing from the upstream to downstream sediments. The biomass proportion of bacterial populations in the midstream sediment showed a significantly difference from that in the other sediments, suggesting that there may be an environmental pressure on the midstream bacterial community. Lorenz curves, together with Gini coefficients were successfully applied to the 2-DGE mapping data for resolving evenness of bacterial populations, and showed that the plotted curve from high-resolution 2-DGE mapping became less linear and more an exponential function than that of the 1-DGE methods such as chain length analysis and denaturing gradient gel electrophoresis, suggesting that the 2-DGE mapping may achieve a more detailed evaluation of bacterial community. In conclusion, the 2-DGE mapping combined with the selective primer pairs enables bacterial community structure analysis in river sediment and thus it can also monitor sediment pollution based on the change of bacterial community structure.

[The use of polymer gel dosimetry to measure dose distribution around metallic implants].

PubMed

Nagahata, Tomomasa; Yamaguchi, Hajime; Monzen, Hajime; Nishimura, Yasumasa

2014-10-01

A semi-solid polymer dosimetry system using agar was developed to measure the dose distribution close to metallic implants. Dosimetry of heterogeneous fields where electron density markedly varies is often problematic. This prompted us to develop a polymer gel dosimetry technique using agar to measure the dose distribution near substance boundaries. Varying the concentration of an oxygen scavenger (tetra-hydroxymethyl phosphonium chloride) showed the absorbed dose and transverse relaxation rate of the magnetic resonance signal to be linear between 3 and 12 Gy. Although a change in the dosimeter due to oxidization was observed in room air after 24 hours, no such effects were observed in the first 4 hours. The dose distribution around the metal implants was measured using agar dosimetry. The metals tested were a lead rod, a titanium hip joint, and a metallic stent. A maximum 30% dose increase was observed near the lead rod, but only a 3% increase in the absorbed dose was noted near the surface of the titanium hip joint and metallic stent. Semi-solid polymer dosimetry using agar thus appears to be a useful method for dosimetry around metallic substances.

Molecular Typing of Clostridium perfringens from a Food-Borne Disease Outbreak in a Nursing Home: Ribotyping versus Pulsed-Field Gel Electrophoresis

PubMed Central

Schalch, Barbara; Bader, Lutz; Schau, Hans-Peter; Bergmann, Rolf; Rometsch, Andrea; Maydl, Gertraud; Keßler, Silvia

2003-01-01

In 1998, 21 inhabitants of a German nursing home fell ill with acute gastroenteritis after consumption of minced beef heart (P. Graf and L. Bader, Epidemiol. Bull. 41:327-329, 2000). Two residents died during hospital treatment. Seventeen Clostridium perfringens strains were collected from two different dishes and from patients' stool samples and autopsy materials. A majority of these isolates was not typeable by restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE). Subsequent ribotyping of C. perfringens distinguished four different groups. The same ribopattern was detected in a minced beef heart dish, in autopsy material from the two deceased patients, and additionally in stool samples from six further residents who had fallen ill with diarrhea. Three further ribopatterns from food and autopsy materials could be differentiated. As chromosomal macrorestriction with subsequent PFGE is generally regarded more useful than ribotyping for molecular strain analysis, four selected isolates were lysed in parallel with a standard protocol and two nucleases inhibiting modifications. Neither of these methods could differentiate all of the isolates. These results suggest that PFGE with the current standard protocols is not able to characterize all C. perfringens isolates from food-borne disease investigations and that ribotyping is still a helpful method for molecular identification of clonal relationships. PMID:12574310

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.

PubMed

Rodrigues, Silas Pessini; Ventura, José Aires; Zingali, R B; Fernandes, P M B

2009-01-01

A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

NASA Astrophysics Data System (ADS)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry

NASA Astrophysics Data System (ADS)

Ogorzalek Loo, Rachel R.; Mitchell, Charles; Stevenson, Tracy I.; Loo, Joseph A.; Andrews, Philip C.

1997-12-01

Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclonal antibody anti-[beta]-galactosidase transferred to Goretex. Polypropylene, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF required that the membrane be kept wet prior to application of matrix. Considerations in mass accuracy for analysis from large-area membranes with continuous extraction and delayed extraction were explored, as were remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping.

Acanthamoeba on Sabouraud's agar from a patient with keratitis

PubMed Central

Baradkar, Vasant; Samal, Badhuli; Mali, Swapna A; Kulkarni, Ketaki; Shastri, Jayanthi

2011-01-01

A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application. PMID:23508061

Simplified transient isotachophoresis/capillary gel electrophoresis method for highly sensitive analysis of polymerase chain reaction samples on a microchip with laser-induced fluorescence detection.

PubMed

Liu, Dayu; Ou, Ziyou; Xu, Mingfei; Wang, Lihui

2008-12-19

We present a sensitive, simple and robust on-chip transient isotachophoresis/capillary gel electrophoresis (tITP/CGE) method for the analysis of polymerase chain reaction (PCR) samples. Using chloride ions in the PCR buffer and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the background electrolyte, respectively, as the leading and terminating electrolytes, the tITP preconcentration was coupled with CGE separation with double-T shaped channel network. The tITP/CGE separation was carried out with a single running buffer. The separation process involved only two steps that were performed continuously with the sequential switching of four voltage outputs. The tITP/CGE method showed an analysis time and a separation efficiency comparable to those of standard CGE, while the signal intensity was enhanced by factors of over 20. The limit of detection of the chip-based tITP/CGE method was estimated to be 1.1 ng/mL of DNA in 1x PCR buffer using confocal fluorescence detection following 473 nm laser excitation.

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese.

PubMed

Henri-Dubernet, Ségolène; Desmasures, Nathalie; Guéguen, Micheline

2008-03-01

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.

Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

PubMed Central

2010-01-01

Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M). Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture. Conclusions This work reports

Observation of laser-induced elastic waves in agar skin phantoms using a high-speed camera and a laser-beam-deflection probe

PubMed Central

Laloš, Jernej; Gregorčič, Peter; Jezeršek, Matija

2018-01-01

We present an optical study of elastic wave propagation inside skin phantoms consisting of agar gel as induced by an Er:YAG (wavelength of 2.94 μm) laser pulse. A laser-beam-deflection probe is used to measure ultrasonic propagation and a high-speed camera is used to record displacements in ablation-induced elastic transients. These measurements are further analyzed with a custom developed image recognition algorithm utilizing the methods of particle image velocimetry and spline interpolation to determine point trajectories, material displacement and strain during the passing of the transients. The results indicate that the ablation-induced elastic waves propagate with a velocity of 1 m/s and amplitudes of 0.1 mm. Compared to them, the measured velocities of ultrasonic waves are much higher, within the range of 1.42–1.51 km/s, while their amplitudes are three orders of magnitude smaller. This proves that the agar gel may be used as a rudimental skin and soft tissue substitute in biomedical research, since its polymeric structure reproduces adequate soft-solid properties and its transparency for visible light makes it convenient to study with optical instruments. The results presented provide an insight into the distribution of laser-induced elastic transients in soft tissue phantoms, while the experimental approach serves as a foundation for further research of laser-induced mechanical effects deeper in the tissue. PMID:29675327

Observation of laser-induced elastic waves in agar skin phantoms using a high-speed camera and a laser-beam-deflection probe.

PubMed

Laloš, Jernej; Gregorčič, Peter; Jezeršek, Matija

2018-04-01

We present an optical study of elastic wave propagation inside skin phantoms consisting of agar gel as induced by an Er:YAG (wavelength of 2.94 μm) laser pulse. A laser-beam-deflection probe is used to measure ultrasonic propagation and a high-speed camera is used to record displacements in ablation-induced elastic transients. These measurements are further analyzed with a custom developed image recognition algorithm utilizing the methods of particle image velocimetry and spline interpolation to determine point trajectories, material displacement and strain during the passing of the transients. The results indicate that the ablation-induced elastic waves propagate with a velocity of 1 m/s and amplitudes of 0.1 mm. Compared to them, the measured velocities of ultrasonic waves are much higher, within the range of 1.42-1.51 km/s, while their amplitudes are three orders of magnitude smaller. This proves that the agar gel may be used as a rudimental skin and soft tissue substitute in biomedical research, since its polymeric structure reproduces adequate soft-solid properties and its transparency for visible light makes it convenient to study with optical instruments. The results presented provide an insight into the distribution of laser-induced elastic transients in soft tissue phantoms, while the experimental approach serves as a foundation for further research of laser-induced mechanical effects deeper in the tissue.

Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus.

PubMed

Nithya, Sai; Nimal, T R; Baranwal, Gaurav; Suresh, Maneesha K; C P, Anju; Anil Kumar, V; Gopi Mohan, C; Jayakumar, R; Biswas, Raja

2018-04-15

Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic interactions of LST with chitosan were studied by molecular docking studies. The rheology and the antibacterial properties of the developed LSTC gel were evaluated. The developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus survival by ∼3 Log 10 CFU/mL in the presence of LSTC gel. The cytocompatibility of the gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We conclude that LSTC gel could be highly effectual and advantageous over antibiotics in treating staphylococcal-topical and biofilm infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

PubMed

Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

2009-09-01

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

DOEpatents

Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

2012-12-11

Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

Microfluidic device having an immobilized pH gradient and page gels for protein separation and analysis

DOEpatents

Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

2014-05-20

Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wahl, Karen L.; Colburn, Heather A.; Wunschel, David S.

2010-02-15

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MSn) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or non-irradiated and notmore » in the spores grown in broth. A sample containing approximately 108 spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only 3 false negatives for samples that were below the detection level of the method as documented.« less

Thermal characterization of magnetically aligned carbonyl iron/agar composites.

PubMed

Diaz-Bleis, D; Vales-Pinzón, C; Freile-Pelegrín, Y; Alvarado-Gil, J J

2014-01-01

Composites of magnetic particles into polymeric matrices have received increasing research interest due to their capacity to respond to external magnetic or electromagnetic fields. In this study, agar from Gelidium robustum has been chosen as natural biocompatible polymer to build the matrix of the magnetic carbonyl iron particles (CIP) for their uses in biomedical fields. Heat transfer behavior of the CIP-agar composites containing different concentrations (5, 10, 15, 20, 25 and 30% w/w) of magnetically aligned and non-aligned CIP in the agar matrix was studied using photothermal radiometry (PTR) in the back-propagation emission configuration. The morphology of the CIP-agar composites with aligned and non-aligned CIP under magnetic field was also evaluated by scanning electron microscopy (SEM). The results revealed a dominant effect of CIP concentration over the alignment patterns induced by the magnetic field, which agrees with the behavior of the thermal diffusivity and thermal conductivity. Agar served as a perfect matrix to be used with CIP, and CIP-agar composites magnetically aligned at 20% CIP concentration can be considered as promising 'smart' material for hyperthermia treatments in the biomedical field. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chocolate agar, a differential medium for gram-positive cocci.

PubMed Central

Gunn, B A

1984-01-01

Reactions incurred on chocolate agar by gram-positive cocci were correlated with species identity. Darkening and clearing of the medium was usually associated with the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus simulans, and Streptococcus faecalis. Yellowing of chocolate agar was associated with alpha-hemolytic species of Streptococcus. The study demonstrated that reactions occurring on chocolate agar are useful in identifying gram-positive cocci. PMID:6490866

Antibacterial Activity of Copaiba Oil Gel on Dental Biofilm

PubMed Central

Simões, Cláudia A.C.G.; Conde, Nikeila C. de Oliveira; Venâncio, Gisely N.; Milério, Patrícia S.L.L.; Bandeira, Maria F.C.L.; da Veiga Júnior, Valdir F.

2016-01-01

Amazonian biodiversity products that have been used for years in folk medicine, have emerged as feasible and promising alternatives for the inhibition of microorganisms in dental biofilm. Copaiba oil, a phytotherapic agent widely used by the Amazonian populations, is known for its antibacterial, anti-inflammatory, anesthetic, healing and antitumor medicinal properties. Objective: The aim of this study was to evaluate the in vitro antibacterial activity of copaiba oil (Copaifera multijuga) gel against strains of Streptococcus sp present in dental biofilm. Materials and Methods: The copaiba oil was obtained and the chemical components were identified. The oil emulsions were formulated and used with the Brain Heart Infusion agar diffusion method with strains of Streptococcus mitis, Streptococcus constellatus and Streptococcus salivarius isolated from patients as well as standard strains of S. mitis (ATCC903), S. mutans (ATCC10449), S. sanguinis (ATCC15300) and S. oralis (ATCC10557). The study groups were as follows: experimental copaiba oil gel, 1% chlorhexidine gel (positive control) and base gel (negative control). The seeded plates were incubated at 37ºC for 12, 24 and 48 hours, respectively. The results obtained were analyzed by Shapiro-Wilk and Friedman Tests (p<0.05) for non parametric data and the Tukey test was used for pH values with 5% level of significance. Results: The experimental copaiba oil gel and 1% chlorhexidine gel showed antibacterial activity against the tested microorganisms. Conclusion: The copaiba oil gel demonstrated antibacterial activity against all the strains of Streptococcus sp tested, suggesting that it can be used for dental biofilm control. PMID:27386004

[Analysis of bactericidal material generated by electrical devices advertising bactericidal ability against bacteria on the agar gel plates].

PubMed

Nishimura, Hidekazu

2012-11-01

Several Japanese companies sell electrical devices advertised as effective in inactivating viruses and killing bacteria by releasing special materials, e.g., Plasmacluster ions, Nanoe particle and minus ions, into the air. These companies claim that their devices killed bacteria on plates in their own experiments. We tested device effectiveness using the same experiments from the Plasmacluster ioniser SHARP Co., Japan, the Nanoe generator Panasonic Co., Japan, and the Vion KING JIM Co., Japan, to test their advertising claims. Bactericidal ability on agar plate was tested, using Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Enterococcus faecalis as follows: the medium containing a certain amount of each bacterium was put onto an agar plate and smeared. Plates were kept in a closed chamber (inner volume 14.4 m3) or a glove box (inner volume 0.2 m), with one of the devices run for 2 hours. Plates not exposed to any device were used as controls. Each plate was retrieved and put in an incubator to count the number of bacterial colonies formed on the plate. There was no significant difference in the number of colonies on plates exposed to devices compared to control, in the number for all devices, or in all bacteria tested in experiments in the 14.4 m3 chamber. These results strongly suggest that these devices have almost no bactericidal effect, at least in space exceeding this volume. Colony formation was suppressed in the glove box in all devices and in all bacteria tested except P. aeruginosa, although the degree of suppression differed among experiments. The colony formation suppression mechanism was analyzed, and indicated that:colony formation did not change even after the removal of Plasmacluster ions, Nanoe particles, or negative ions from the air, while colony formation was decreased drastically by the removal of ozone from space, which was revealed to be generated inevitably during device operation. These results strongly suggest that the

Integrated microfluidic chip for rapid DNA digestion and time-resolved capillary electrophoresis analysis

PubMed Central

Lin, Che-Hsin; Wang, Yao-Nan; Fu, Lung-Ming

2012-01-01

An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications. PMID:22662085

Protein Electrophoresis/Immunofixation Electrophoresis

MedlinePlus

... underlying disease. Follow-up tests may include, for example, albumin , immunoelectrophoresis, serum free light chains , quantitative immunoglobuins , ... the disease and the effectiveness of treatment. Some examples of when an electrophoresis test may be ordered ...

Sol gel derived hydroxyapatite coatings on titanium and its alloy Ti6Al4V

NASA Astrophysics Data System (ADS)

Stoch, A.; Jastrzebski, W.; Długoń, E.; Lejda, W.; Trybalska, B.; Stoch, G. J.; Adamczyk, A.

2005-06-01

Titanium has been used for many medical and dental applications; however, its joining to a living bone is not satisfactorily good or the implant integration with bone tissue takes several months.The aim of this work is to produce hydroxyapatite (HAP) coatings on titanium and its alloy for facilitating and shortening the processes towards osseointegration. HAP coatings were obtained by sol-gel method with sol solutions prepared from calcium nitrate tetrahydrate and triammonium phosphate trihydrate as the calcium and phosphorous sources. Two types of gelatine were added to the sol: agar-agar or animals gelatine. Both were found to enhance the formation and stability of amorphous HAP using soluble salts as the sources of calcium and phosphate. HAP coatings were deposited from HAP-GEL sol using dip-withdrawal technique, then the plates were dried and annealed at temperatures 460-750 °C. FTIR spectroscopy and XRD analysis were used to study the phase composition of phosphate coatings. Morphology and chemical analysis of HAP layers was performed using a scanning electron microscope equipped with an energy dispersive X-ray analyser (SEM+EDX). The biological activity of sol-gel phosphate coatings was observed during thermostatic held in simulated body fluid (SBF). It was found that chemical composition and structure of HAP coatings depends on pH and final thermal treatment of the layer.

Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE).

PubMed

Hoppe-Seyler, T S; Jaeger, B; Bockelmann, W; Noordman, W H; Geis, A; Heller, K J

2003-09-01

ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

PubMed

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-06-23

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.

PubMed

Deshmukh, Ameya A; Weist, Jessica L; Leight, Jennifer L

2018-05-01

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

Factors Affecting Selectivity of Brilliant Green-Phenol Red Agar for Salmonellae

PubMed Central

Moats, W. A.; Kinner, J. A.

1974-01-01

Commercial brilliant green (BG)-sulfa agar was found to be nonselective toward a test series of Enterobacteriaceae. Various formulations of BG were prepared by using Trypticase soy agar (BBL) as a base. Results were more reproducible when BG dye was added after sterilization than before. Sulfonamides improved selectivity as compared with brilliant green alone. Sulfanilamide (SN) was slightly more selective for salmonellae than other sulfonamides tested. Bile salts and sodium dodecyl sulfate markedly reduced the toxicity of BG to all the test bacteria. Enterobacter strains were most difficult to inhibit. A combination of 5 mg of BG and 1 g of SN/liter prevented growth of Proteus mirabilis and Escherichia coli and retarded growth of Enterobacter strains. The BG-SN agars were superior in selectivity to a series of commercial agars tested, and numbers of salmonellae recovered on BG-SN agar and Trypticase soy agar (BBL) were the same. Brilliant green agars with various degrees of selectivity are described. PMID:4589120

Genetic Characterization of Atypical Enteropathogenic Escherichia coli Isolates from Ewes' Milk, Sheep Farm Environments, and Humans by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

PubMed Central

Otero, Verónica; Rodríguez-Calleja, José-María; Otero, Andrés; García-López, María-Luisa

2013-01-01

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water. PMID:23872571

Protein Profile Analysis of Two Australian Snake Venoms by One- Dimensional Gel Electrophoresis and MS/MS Experiments.

PubMed

Georgieva, Dessislava; Hildebrand, Diana; Simas, Rodrigo; Coronado, Monika A; Kwiatkowski, Marcel; Schlüter, Hartmut; Arni, Raghuvir; Spencer, Patrick; Betzel, Christian

2017-01-01

The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility.

PubMed

Mullane, N R; Whyte, P; Wall, P G; Quinn, T; Fanning, S

2007-05-01

Enterobacter sakazakii (E. sakazakii) contamination of powdered infant formula (PIF) and its processing environment was monitored between April 2005 and March 2006. The purpose of the monitoring programme was to locate points of contamination, investigate clonal persistence, and identify possible dissemination routes along the processing chain. A total of 80 E. sakazakii isolates were recovered from the manufacturing facility. The overall frequency of isolation of E. sakazakii in intermediate and final product was 2.5%, while specific locations in the processing environment were contaminated at frequencies up to 31%. All E. sakazakii isolates were characterised by pulsed-field gel electrophoresis (PFGE). XbaI macrorestriction digests yielded 19 unique pulse-types that could be grouped into 6 clusters of between 5 and 32 isolates. The formation of large clusters was consistent with the presence of a number of clones in the manufacturing environment. While the majority of isolates were of environmental origin (72.5%), no cluster was confined to one specific location and indistinguishable PFGE profiles were generated from isolates cultured from the manufacturing environment, sampling points along the processing chain and from intermediate and final product. These findings suggest that the manufacturing environment serves as a key route for sporadic contamination of PIF. These data will support the development of efficient intervention measures contributing to the reduction of E. sakazakii in the PIF processing chain.

Kidney Cell Electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

PubMed

Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

2014-08-01

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize.

PubMed

Sánchez, Laura; González, Ramón; Crego, Antonio L; Cifuentes, Alejandro

2007-03-01

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.

Compositions of maple sap microflora and collection system biofilms evaluated by scanning electron microscopy and denaturing gradient gel electrophoresis.

PubMed

Lagacé, L; Jacques, M; Mafu, A A; Roy, D

2006-05-25

The bacterial microflora of maple sap and biofilms in collection system tubing were studied through the use of bacterial counts, scanning electron microscopy (SEM) of surfaces and the analysis of 16S rRNA gene by denaturing gradient gel electrophoresis (DGGE). Samples were taken at five times during the 2002 and 2003 seasons in order to follow the changes in the microflora of this complex ecosystem. Bacterial counts showed the growth of bacterial populations as the season advanced. These populations were mainly composed of psychrotrophic bacteria and Pseudomonas spp. SEM results confirmed the suspected presence of biofilms on the inner surfaces of tubing samples. Bacterial colonization and biofilm formation progressively increased during the season for both lateral and main line surfaces, and biofilms were mainly composed of rod shape bacteria. The bacterial microflora profiles obtained for sap and corresponding biofilm by DGGE showed up to 12 major bands. The Shannon-Weaver index of diversity (H) calculated from DGGE bands were statistically higher for sap samples compared to biofilm. The diversity index was relatively stable or increasing for lateral line sap and biofilm samples during the season while the diversity index for sap and biofilm samples of the main line showed a decreasing profile as the season progressed. Sequence analysis of major DGGE bands revealed the predominance of bacteria from the genera Pseudomonas, Rahnella and another, unidentified genus. The results describe the composition of sap collection system microflora as well as the formation of biofilms and will be useful for further studies on factors affecting maple product quality.

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

PubMed Central

Smalla, K.; Wieland, G.; Buchner, A.; Zock, A.; Parzy, J.; Kaiser, S.; Roskot, N.; Heuer, H.; Berg, G.

2001-01-01

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands. PMID:11571180

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

PubMed

Sowmithra, K; Shetty, N J; Jha, S K; Chaubey, R C

2015-12-01

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species

EPA Science Inventory