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Sample records for native polyacrylamide gel

  1. Resolving mitochondrial protein complexes using non-gradient blue native polyacrylamide gel electrophoresis

    PubMed Central

    Yan, Liang-Jun; Forster, Michael J.

    2009-01-01

    Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomic analysis of high molecular weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this paper, we present an alternative BN-PAGE method that uses highly porous, non-gradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I-V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be simultaneously detected on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, non-gradient gel electrophoretic approach that should be useful in functional proteomics studies. PMID:19348780

  2. Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE).

    PubMed

    Jha, Pooja; Wang, Xu; Auwerx, Johan

    2016-01-01

    Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in-gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN-PAGE). © 2016 by John Wiley & Sons, Inc. PMID:26928661

  3. A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.

    PubMed

    Ladig, Roman; Sommer, Maik S; Hahn, Alexander; Leisegang, Matthias S; Papasotiriou, Dimitrios G; Ibrahim, Mohamed; Elkehal, Rajae; Karas, Michael; Zickermann, Volker; Gutensohn, Michael; Brandt, Ulrich; Klösgen, Ralf Bernd; Schleiff, Enrico

    2011-07-01

    Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons. PMID:21418111

  4. Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

    PubMed Central

    Tang, Shuiquan; Chan, Winnie W. M.; Fletcher, Kelly E.; Seifert, Jana; Liang, Xiaoming; Lffler, Frank E.; Adrian, Lorenz

    2013-01-01

    Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family. PMID:23204411

  5. A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    PubMed

    Pristov, Jelena Bogdanović; Opačić, Miloš; Dimitrijević, Milena; Babić, Nikolina; Spasojević, Ivan

    2015-07-01

    We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation-emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide. PMID:25862081

  6. Electroblotting from Polyacrylamide Gels.

    PubMed

    Goldman, Aaron; Ursitti, Jeanine A; Mozdzanowski, Jacek; Speicher, David W

    2015-01-01

    Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. 2015 by John Wiley & Sons, Inc. PMID:26521711

  7. Polyacrylamide gel electrophoresis.

    PubMed

    Chrambach, A; Rodbard, D

    1971-04-30

    Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0 degrees and 25 degrees C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities. PMID:4927678

  8. Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis.

    PubMed

    Sabar, Mohammed; Balk, Janneke; Leaver, Christopher J

    2005-12-01

    Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F(1)F(0)-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed. PMID:16297078

  9. Silver staining techniques of polyacrylamide gels.

    PubMed

    Bartsch, Holger; Arndt, Claudia; Koristka, Stefanie; Cartellieri, Marc; Bachmann, Michael

    2012-01-01

    Although the main application for polyacrylamide gels is the separation and subsequent blotting of proteins for immunodetection, there are tasks that need staining of proteins in the polyacrylamide gel. Several different staining techniques exist for protein staining in SDS gels that differ in their sensitivity, their expenditure of time, and other aspects. Still, silver staining is the most sensitive and reliable staining technique. Because this technique was developed in the 1970s, a huge number of variations exist. Therefore, we will provide herein three methods, which are robust and easy to perform. PMID:22585513

  10. 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

  11. Visible fluorescent detection of proteins in polyacrylamide gels without staining.

    PubMed

    Ladner, Carol L; Yang, Jing; Turner, Raymond J; Edwards, Robert A

    2004-03-01

    2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography. PMID:14769330

  12. Polyacrylamide Gel Electrophoresis for Purification of Large Amounts of RNA.

    PubMed

    Meyer, Mélanie; Masquida, Benoît

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) constitutes a powerful technique for the efficient purification of RNA molecules dedicated to applications that require high purity levels. PAGE allows for the fractionation of RNA obtained from cell extracts, chemical or enzymatic synthesis, or modification experiments. Native or denaturing conditions can be chosen for analytical or preparative-scale separations and the nucleotide resolution can be tuned by changing the percentage and reticulation of the gel material. In this protocol, we focus on the preparation of milligram-scale amounts of ~200 nucleotides (nt) RNA molecules that were used in subsequent crystallization experiments. PMID:26227037

  13. Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis.

    PubMed

    Trudel, J; Asselin, A

    1990-09-01

    Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis. PMID:2281870

  14. Silver staining of proteins in polyacrylamide gels.

    PubMed

    Chevallet, Mireille; Luche, Sylvie; Rabilloud, Thierry

    2006-01-01

    Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks. PMID:17487168

  15. Micro-size polyacrylamide gel electrophoresis system

    NASA Astrophysics Data System (ADS)

    Hinson, W. G.; Pipkin, J. L.; Anson, J. F.; Casciano, D. A.; Burns, E. R.

    1987-09-01

    The development and characterization of a micro-size two-dimensional polyacrylamide gel electrophoresis system is described. Some of the techniques which have evolved with use of the system are also discussed. This apparatus has unique features which provide advantages over other small scale units. Up to ten first- and second-dimension gels can be processed simultaneously with excellent resolution of protein regions. Consistent reproducibility is possible from protein samples as small as 400 ng and individual protein regions as small as 1 pg can be visualized by silver staining of the two-dimensional gels. Similar sensitivities are achieved in autoradiographs of 3H-labeled proteins extracted from the nuclei of cultured cells. The application of this system in conjunction with flow cytometric examination of nuclear DNA and electrostatic cell sorting of specific cell nuclei to provide homogeneous sample populations, allows subtle variations in isotope incorporation in proteins to be detected; whereas many times in generalized tissue samples these changes are masked. Also, these techniques elucidate the effects of external stimuli (chemicals, drugs, or environment) on protein synthesis and phosphorylation for analyses and comparison. Fabrication drawings are available upon request.

  16. Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

    PubMed Central

    2011-01-01

    Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work. Conclusions In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction. PMID:21241518

  17. Eosin Y staining of proteins in polyacrylamide gels.

    PubMed

    Lin, F; Fan, W; Wise, G E

    1991-08-01

    A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins. PMID:1723249

  18. 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

  19. The instantaneous monitoring of polyacrylamide gels during electrophoresis.

    PubMed Central

    Elliott, A

    1976-01-01

    The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel. Images PLATE 1 PLATE 2 PMID:1008832

  20. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

    SciTech Connect

    Lacks, S.A.; Springhorn, S.S.

    1980-08-10

    A number of enzymes, including amylases, dehydrogenases, and proteases, were shown to be renaturable after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Enzyme activity was detected in situ by action on substrates introduced into the gel and subsequent staining of either the product or unreacted substrate. Enzymes appeared to recover activity as soon as the detergent diffused out of the gel. Renatured enzymes were retained in gels after electrophoresis longer than native enzymes which had been subjected to electrophoresis in the absence of detergent. Re-electrophoresis of the renatured enzymes showed that part of the retained activity was physically anchored to the gel, possibly by the folding of polypeptides around the gel matrix as the enzymes were renatured.

  1. The electrophoresis of transferrins in urea/polyacrylamide gels.

    PubMed Central

    Evans, R W; Williams, J

    1980-01-01

    The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss of iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. PMID:7213345

  2. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis

    PubMed Central

    2014-01-01

    Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications. PMID:24735559

  3. Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

    SciTech Connect

    Longley, M.J.; Mosbaugh, D.W. )

    1991-03-12

    The authors have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined {sup 32}P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their ({sup 32}P)DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase {beta} were found to be similar under both in vitro and in situ conditions. With 3{prime}-terminally matched and mismatched ({sup 32}P)DNA substrates in the same activity gel, DNA polymerase and/or 3{prime} to 5{prime} exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. Several restriction endonucleases and the tripeptide (Lys-Try-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.

  4. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

    PubMed Central

    Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution. PMID:26650843

  5. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    NASA Technical Reports Server (NTRS)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  6. A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis.

    PubMed

    Krisman, C R; Blumenfeld, M L

    1986-05-01

    A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures. PMID:2425655

  7. Glutamine Synthetase Regulation, Adenylylation State, and Strain Specificity Analyzed by Polyacrylamide Gel Electrophoresis

    PubMed Central

    Bender, Robert A.; Streicher, Stanley L.

    1979-01-01

    We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GSE) and Klebsiella aerogenes (GSK). In gels containing sodium dodecyl sulfate (SDS), we found that GSK had a mobility which differed significantly from that of GSE. In addition, for both GSK and GSE, adenylylated subunits (GSK-adenosine 5′-monophosphate [AMP] and GSE-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GSK-AMP < GSK < GSE-AMP < GSE. We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GSK, and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GSK or GSK-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele. Images PMID:33958

  8. Electrophoresis of Dyes and Proteins in Poly(Acrylamide) Gel Containing Immobilized Bilayer Membranes

    NASA Astrophysics Data System (ADS)

    Ishihara, Hiroki; Matsuo, Goh; Sasaki, Takanori; Saito, Yuko; Demura, Makoto; Tsujii, Kaoru

    Electrophoresis of dye stuffs and proteins in poly(acrylamide) gel containing immobilized bilayer membranes have been studied. Bilayer membranes of a polymerizable surfactant, dodecylglyceryl itaconate (DGI), can be immobilized in poly(acrylamide) gels, and the hybrid gels are first applied to a substrate of the poly(acrylamide) gel electrophoresis (PAGE). The bilayer-membranes-immobilized-gel (abbreviated as BM-gel) showed different separation behaviors from those by the conventional PAGE. The separation behavior of dye stuffs suggests that the bilayer membranes in the BM-gel work as a separator of the test molecules due to their hydrophilic/hydrophobic nature. Water-soluble proteins migrated faster in the BM-gels than in the simple poly(acrylamide) gels. Membrane proteins, on the other hand, did not move at all in the BM-gels probably because the protein molecules were entrapped firmly inside the bilayer membranes.

  9. Physical properties of polyacrylamide gels probed by AFM and rheology

    NASA Astrophysics Data System (ADS)

    Abidine, Yara; Laurent, Valérie M.; Michel, Richard; Duperray, Alain; Iulian Palade, Liviu; Verdier, Claude

    2015-02-01

    Polymer gels have been shown to behave as viscoelastic materials but only a small amount of data is usually provided in the glass transition. In this paper, the dynamic moduli G\\prime and G\\prime\\prime of polyacrylamide hydrogels are investigated using both an AFM in contact force modulation mode and a classical rheometer. The validity is shown by the matching of the two techniques. Measurements are carried out on gels of increasing polymer concentration in a wide frequency range. A model based on fractional derivatives is successfully used, covering the whole frequency range. G\\text{N}0 , the plateau modulus, as well as several other parameters are obtained at low frequencies. The model also predicts the slope a of both moduli in the glass transition, and a transition frequency f\\text{T} is introduced to separate the gel-like behavior with the glassy state. Its variation with polymer content c gives a dependence f\\text{T}∼ c1.6 , in good agreement with previous theories. Therefore, the AFM data provides new information on the physics of polymer gels.

  10. Characteristics of polyacrylamide gel with THPC and Turnbull Blue gel dosimeters evaluated using optical tomography

    NASA Astrophysics Data System (ADS)

    Pilařová (Vávrů), Kateřina; Kozubíková, Petra; Šolc, Jaroslav; Spěváček, Václav

    2014-11-01

    The purpose of this study was to compare characteristics of radiochromic gel - Turnbull Blue gel (TB gel) with polymer gel - polyacrylamide gel and tetrakis hydroxymethyl phosphonium chloride (PAGAT) using optical tomography. Both types of gels were examined in terms of dose sensitivity, dose response linearity and background value of spectrophotometric absorbance. The calibration curve was obtained for 60Co irradiation performed on Gammacell 220 at predefined gamma dose levels between 0 and 140 Gy for TBG and 0-15 Gy for PAGAT. To measure relative dose distributions from stereotactic irradiation, dosimeters were irradiated on Leksell Gamma Knife Perfexion. The cylindrical glass housings filled with gel were attached to the stereotactic frame. They were exposed with single shot and 16 mm collimator by 65 Gy to a 50% prescription isodose for TB gel and 4 Gy to a 50% prescription isodose for PAGAT. Evaluations of dosimeters were performed on an UV-vis Spectrophotometer Helios β and an optical cone beam homemade tomography scanner with a 16-bit astronomy CCD camera with a set of color filters. The advantages and potential disadvantages for both types of gel dosimeters were summarized. Dose distribution in central slice and measured profiles of 16 mm shot shows excellent correspondence with treatment planning system Leksell GammaPlan® for both PAGAT and Turnbull Blue gels. Gel dosimeters are suitable for steep dose gradient verification. An optical tomography evaluation method is successful. Dose response characteristics of TB gel and PAGAT gel are presented.

  11. Scalable lithography from Natural DNA Patterns via polyacrylamide gel.

    PubMed

    Qu, JieHao; Hou, XianLiang; Fan, WanChao; Xi, GuangHui; Diao, HongYan; Liu, XiangDon

    2015-01-01

    A facile strategy for fabricating scalable stamps has been developed using cross-linked polyacrylamide gel (PAMG) that controllably and precisely shrinks and swells with water content. Aligned patterns of natural DNA molecules were prepared by evaporative self-assembly on a PMMA substrate, and were transferred to unsaturated polyester resin (UPR) to form a negative replica. The negative was used to pattern the linear structures onto the surface of water-swollen PAMG, and the pattern sizes on the PAMG stamp were customized by adjusting the water content of the PAMG. As a result, consistent reproduction of DNA patterns could be achieved with feature sizes that can be controlled over the range of 40%-200% of the original pattern dimensions. This methodology is novel and may pave a new avenue for manufacturing stamp-based functional nanostructures in a simple and cost-effective manner on a large scale. PMID:26639572

  12. Scalable lithography from Natural DNA Patterns via polyacrylamide gel

    PubMed Central

    Qu, JieHao; Hou, XianLiang; Fan, WanChao; Xi, GuangHui; Diao, HongYan; Liu, XiangDon

    2015-01-01

    A facile strategy for fabricating scalable stamps has been developed using cross-linked polyacrylamide gel (PAMG) that controllably and precisely shrinks and swells with water content. Aligned patterns of natural DNA molecules were prepared by evaporative self-assembly on a PMMA substrate, and were transferred to unsaturated polyester resin (UPR) to form a negative replica. The negative was used to pattern the linear structures onto the surface of water-swollen PAMG, and the pattern sizes on the PAMG stamp were customized by adjusting the water content of the PAMG. As a result, consistent reproduction of DNA patterns could be achieved with feature sizes that can be controlled over the range of 40%–200% of the original pattern dimensions. This methodology is novel and may pave a new avenue for manufacturing stamp-based functional nanostructures in a simple and cost-effective manner on a large scale. PMID:26639572

  13. Scalable lithography from Natural DNA Patterns via polyacrylamide gel

    NASA Astrophysics Data System (ADS)

    Qu, Jiehao; Hou, Xianliang; Fan, Wanchao; Xi, Guanghui; Diao, Hongyan; Liu, Xiangdon

    2015-12-01

    A facile strategy for fabricating scalable stamps has been developed using cross-linked polyacrylamide gel (PAMG) that controllably and precisely shrinks and swells with water content. Aligned patterns of natural DNA molecules were prepared by evaporative self-assembly on a PMMA substrate, and were transferred to unsaturated polyester resin (UPR) to form a negative replica. The negative was used to pattern the linear structures onto the surface of water-swollen PAMG, and the pattern sizes on the PAMG stamp were customized by adjusting the water content of the PAMG. As a result, consistent reproduction of DNA patterns could be achieved with feature sizes that can be controlled over the range of 40%-200% of the original pattern dimensions. This methodology is novel and may pave a new avenue for manufacturing stamp-based functional nanostructures in a simple and cost-effective manner on a large scale.

  14. Rheological properties of reactive extrusion modified waxy starch and waxy starch-polyacrylamide copolymer gels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rheological properties of modified waxy starch and waxy starch-polyacrylamide graft copolymers prepared by reactive extrusion were investigated. Both materials can absorb huge amount of water and form gels. The modified waxy starch and waxy starch-polyacrylamide graft copolymer gels all exhibite...

  15. A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

    PubMed

    Leong, L M; Tan, B H; Ho, K K

    1992-12-01

    Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well. PMID:1282787

  16. Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase.

    PubMed

    Yang, Jie; Yang, Xiaodan; Ye, Xiuyun; Lin, Juan

    2016-01-15

    An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly. PMID:26475566

  17. Peptide mapping of basic proteins by proteolysis in acetic acid/urea-minislab polyacrylamide gels.

    PubMed

    Davie, J R

    1985-02-01

    A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications. PMID:3887981

  18. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis.

    PubMed Central

    Appel, R D; Sanchez, J C; Bairoch, A; Golaz, O; Ravier, F; Pasquali, C; Hughes, G J; Hochstrasser, D F

    1994-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), created and maintained at the University Hospital of Geneva in collaboration with the Department of Medical Biochemistry of Geneva University. The proteins have been identified on various 2-D PAGE reference maps by microsequencing, immunoblotting, gel comparison and amino acid composition. Images PMID:7937063

  19. Effects of degree of hydrolysis and shear on gelation reaction kinetics and gel strength. [Polyacrylamides

    SciTech Connect

    Gao, Hong W.

    1991-02-01

    Gelation tests were conducted to investigate the effect of the degree of hydrolysis on gelation reaction kinetics and gel strength using four low-molecular-weight polyacrylamides (MW = 400,000 daltons), which were 10% (HPAM1-10), 20% (HPAM1-20), 30% (HAPM1-30), and 40% (HPAM-40) hydrolyzed, and Cr-3 (pH = 4.8) and Al-3 (pH = 7.0) crosslinkers. Results showed that for polymer/Cr-3 gel systems, samples prepared with a low-molecular-weight polyacrylamide polymer, which was 20% hydrolyzed, gelled at a faster rate and retained higher gel strength than those prepared with a low-molecular-weight polyacrylamide polymer, which was 10% hydrolyzed. Under the screening condition, no viscosity enhancement was observed in samples prepared with polymers having a degree of hydrolysis equal to or greater than 30%. For polymer/Al-3 gel systems, samples prepared with a low-molecular-weight polyacrylamide polymer, which was 20% hydrolyzed, gelled at the fastest rate and retained the strongest gel strength among the polymer/Al-3 gel systems prepared with four low-molecular-weight polyacrylamide polymers, which were 10, 20, 30, and 40% hydrolyzed, respectively. Gelation tests of gel systems in glass bead packs showed that high shear favored the gelation of a gel system that had a fast rate of gelation, but had an adverse effect on the gelation of three gel systems that had a slow rate of gelation. Weak gels were found to be injectable through porous media. Weak gels were degradable under high shear condition and regained viscosity under low shear conditions. 17 refs., 8 figs., 1 tab.

  20. Optimal parameters for laccase-mediated destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels.

    PubMed

    Yang, Jie; Yang, Xiaodan; Ye, Xiuyun; Lin, Juan

    2016-06-01

    The data presented in this article are related to the research article entitled "Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase" [1]. Laccase is a class of multicopper oxidases that can catalyze oxidation of recalcitrant dyestuffs. This article describes optimal parameters for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R-250, with laccase from basidiomycete Cerrena sp. strain HYB07. Effects of laccase activity, mediator type and concentration, temperature and time on destaining of polyacrylamide gels were evaluated with respect to gel background intensity and protein band signals, and the optimal destaining effects were obtained with 15 U mL(-1) laccase and 2 μM ABTS at 37 °C after 2 h. PMID:26955647

  1. Optimal parameters for laccase-mediated destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels

    PubMed Central

    Yang, Jie; Yang, Xiaodan; Ye, Xiuyun; Lin, Juan

    2016-01-01

    The data presented in this article are related to the research article entitled “Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase” [1]. Laccase is a class of multicopper oxidases that can catalyze oxidation of recalcitrant dyestuffs. This article describes optimal parameters for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R-250, with laccase from basidiomycete Cerrena sp. strain HYB07. Effects of laccase activity, mediator type and concentration, temperature and time on destaining of polyacrylamide gels were evaluated with respect to gel background intensity and protein band signals, and the optimal destaining effects were obtained with 15 U mL−1 laccase and 2 μM ABTS at 37 °C after 2 h. PMID:26955647

  2. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels.

    PubMed

    Lee, C; Levin, A; Branton, D

    1987-11-01

    We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices. PMID:2449094

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

    PubMed

    Hellman, Jukka

    2008-09-01

    Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated. PMID:18607574

  4. Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis.

    PubMed

    Huettel, R N; Dickson, D W; Kaplan, D T

    1983-07-01

    Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes. PMID:19295815

  5. Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

    PubMed

    Wu, D; Regnier, F E

    1992-09-11

    Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved. PMID:1430034

  6. Fluorography of tritium-labeled proteins in silver-stained polyacrylamide gels

    SciTech Connect

    Kulcsar, P.; Prestwich, G.D.

    1988-05-01

    Silver-staining of polyacrylamide gel electrophoresis (PAGE)-separated proteins allows sensitive detection of proteins but severely reduces the ability to detect weak beta-emitters present in the protein band. A simple procedure is described in which silver can be removed from a silver-stained PAGE gel (deargentation) using photographic fixer, and the silver-free gel can be enhanced and used for fluorography. A quantitative study of sensitivity is reported for /sup 3/H-labeled bovine serum albumin with a one-dimensional sodium dodecyl sulfate-PAGE slab gel.

  7. Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Timofeev, E.; Mirzabekov, A.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology

    1998-05-15

    Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino-DNA and amino-oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

  8. Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A-gold.

    PubMed

    Perides, G; Plagens, U; Traub, P

    1986-01-01

    The method of electrophoretically transferring proteins from fixed and stained polyacrylamide gels onto nitrocellulose paper has been reevaluated. It is shown that the tedious destaining of gels is not necessary because Coomassie brilliant blue, although it binds tenaciously to nitrocellulose paper, does not reduce the transfer efficiency of proteins. However, its presence impairs the visibility of proteins as detected, for instance, by the immunogold technique. Therefore, a rapid method for the complete removal of the stain from the nitrocellulose paper after completion of the immunogold procedure was developed. Furthermore, it is shown that proteins from dried polyacrylamide gels can still be transferred onto nitrocellulose sheets with an efficiency of approximately 50% compared to proteins transferred from fixed gels. PMID:2420231

  9. Specific antisera produced by direct immunization with slices of polyacrylamide gel containing small amounts of protein.

    PubMed

    Boulard, C; Lecroisey, A

    1982-01-01

    Rabbits were injected with slices of polyacrylamide gels containing entrapped insect proteins after separation by electrophoresis. Specific antibodies were produced independently of the nature of the gel (with or without sodium dodecyl sulphate) and of the staining technique (amido black or Coomassie Blue). The procedure appears to be a rapid and simple method for production of antibodies specific to proteins separated in minute quantities from a complex mixture. PMID:6282975

  10. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995.

    PubMed Central

    Appel, R D; Sanchez, J C; Bairoch, A; Golaz, O; Ravier, F; Pasquali, C; Hughes, G J; Hochstrasser, D F

    1996-01-01

    SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database. PMID:8594575

  11. A method for staining and stabilizing peroxidase activity in polyacrylamide gel electrophoresis.

    PubMed

    Shimoni, M; Reuveni, R

    1988-11-15

    A procedure was developed for a rapid double staining of peroxidase and other proteins in the same polyacrylamide gels using guaiacol and Coomassie blue. The distinguishable colored bands of peroxidase isozymes and proteins are stable for at least 8 months. PMID:2469356

  12. Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.

    PubMed

    Neumann, U; Khalaf, H; Rimpler, M

    1992-10-01

    A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels. PMID:1456419

  13. Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

    SciTech Connect

    Barany, K.; Barany, M.; Giometti, C. S.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago

    1995-04-28

    Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

  14. Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms

    PubMed Central

    2013-01-01

    Introduction The analysis of polyacrylamide gels is currently carried out manually or automatically. In the automatic method, there are limitations related to the acceptable degree of distortion of lane and band continuity. The available software cannot deal satisfactorily with this type of situations. Therefore, the paper presents an original image analysis method devoid of the aforementioned drawbacks. Material This paper examines polyacrylamide gel images from Li-Cor DNA Sequencer 4300S resulting from the use of the electrophoretic separation of DNA fragments. The acquired images have a resolution dependent on the length of the analysed DNA fragments and typically it is MGNG=38061027 pixels. The images are saved in TIFF format with a grayscale resolution of 16 bits/pixel. The presented image analysis method was performed on gel images resulting from the analysis of DNA methylome profiling in plants exposed to drought stress, carried out with the MSAP (Methylation Sensitive Amplification Polymorphism) technique. Results The results of DNA polymorphism analysis were obtained in less than one second for the Intel Core 2 Quad CPU Q9300@2.5GHz, 8GB RAM. In comparison with other known methods, specificity was 0.95, sensitivity = 0.94 and AUC (Area Under Curve) = 0.98. Conclusions It is possible to carry out this method of DNA polymorphism analysis on distorted images of polyacrylamide gels. The method is fully automatic and does not require any operator intervention. Compared with other methods, it produces the best results and the resulting image is easy to interpret. The presented method of measurement is used in the practical analysis of polyacrylamide gels in the Department of Genetics at the University of Silesia in Katowice, Poland. PMID:23835039

  15. Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

    PubMed Central

    Jerrell, Rachel J.; Parekh, Aron

    2015-01-01

    Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the users specific biological and technical needs. PMID:25590238

  16. A simple apparatus for fast and inexpensive recovery of DNA from polyacrylamide gels.

    PubMed

    Harvey, M; Brisson, I; Guérin, S L

    1993-06-01

    A typical elution procedure for DNA should be easy, quick, inexpensive, highly efficient and must provide DNA of good purity and quality to carry out any further enzymatic reactions. Although a number of procedures and apparatus for the specific isolation of DNA from polyacrylamide gels have been described, none possess all these qualities. In this report, we describe the use of a simple apparatus with which DNA molecules can be eluted from polyacrylamide gels with a recovery efficiency of about 90% prior to ethanol precipitation. The quality of these elution products is good enough to allow their use in any further enzymatic reactions. In addition to the fact that more than a dozen individual samples can be eluted and isolated in less than two hours (less than one hour for a single sample), the estimated low cost per electroeluted sample makes it the most affordable and economical elution method available. PMID:8333961

  17. Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels.

    PubMed

    Westermeier, Reiner

    2006-09-01

    In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible. PMID:17031800

  18. A rapid and simplified method for protein silver staining in polyacrylamide gels.

    PubMed

    Zhao, Lijing; Liu, Cong; Sun, Yinpeng; Ban, Liping

    2012-07-01

    Silver staining is widely used to detect protein in polyacrylamide gels when high sensitivity is required. A simple and rapid protocol for silver staining of proteins following PAGE was developed in the present study. The number of steps was reduced compared to conventional protocol by combining fixing, rinsing, and soaking into a single impregnating step, thus achieving detection of proteins in 20 min. The present method is as sensitive as current protocols with the advantage of saving time and costs. PMID:22821490

  19. Preparation of nano-TiO2 powder by polyacrylamide gel method

    NASA Astrophysics Data System (ADS)

    Fan, Xiaowei; Liang, Xiaoping; Guo, Guanqun; Wang, Rongtao; Li, Jianxin

    2009-07-01

    Polyacrylamide gel method is a new successful technique for preparing nanometer metallic oxide materials, such as α-Al2O3, ZnO and ZrO2. TiO2 made by this method, however, has not been reported. In this study, the nano-TiO2 powder was prepared using the novel method with TiCl3 as raw materials, and with acrylamide and N,N'-methylenediacrylamide as crosslinking agent. The TG showed that the optimization sintering process is that: the polyacrylamide gel was dried at 110°C for 2 hour to obtain the xerogel, and then the grinded xerogel was sintered up to 450°C, meanwhile, kept at 250°C, 410°C and 450°C for 1 hour, respectively, to gain TiO2 particles. The grain size of TiO2 particles is smaller than 25 nm. And those particles are spherical approximately and have good distribution because of the hindering effect of space grid structure. Adding SO4 2- in the polyacrylamide gel process can maintain the content of anatase-TiO2 phase at higher sintering temperature. This may be explained that the SO4 2- can form coordination compound with Ti4+. And the coordination compound is prone to generate anatase-TiO2 with the burning out of SO4 2- , in which structure the adjacent two TiO6 hexahedrons share only one oxygen atom.

  20. Affinity gel electrophoresis of nucleic acids. Specific base- and shape-selective separation of DNA and RNA on polyacrylamide-nucleobase conjugated gel.

    PubMed

    Yashima, E; Suehiro, N; Miyauchi, N; Akashi, M

    1993-11-12

    Two types of affinity gels consisting of cross-linked polyacrylamide and affinity ligands possessing nucleic acid bases were prepared. One type of gel was polyacrylamide-poly(vinylnucleobase) conjugated gel, where the poly(vinylnucleobase) such as poly(9-vinyladenine) (PVAd) bearing a nucleobase in the side-chain was entrapped in the gel matrix. The other type of gel, in which a nucleobase such as adenine is chemically bonded to polyacrylamide gel, was prepared by copolymerization of acrylamide, cross-linker and 9-vinyladenine. These affinity gels, especially the former, demonstrated characteristic nucleobase- and shape-selective separation of nucleic acids. The gels showed high affinity for single-stranded DNA and both single- and double-stranded polynucleotides and could separate a double-stranded DNA in mixtures of double-stranded DNA and polynucleotides. The electrophoretic mobilities of poly(uridylic acid) and poly(inosinic acid) were selectively retarded even in the presence of 7 M urea. The electrophoretic behaviours of nucleic acids on the polyacrylamide-PVAd conjugated gels were compared with those on the agarose-PVAd conjugated gel. The effects of urea, temperature and concentration of PVAd were also examined. The polyacrylamide-PVAd conjugated gel served to elucidate interactions between PVAd and nucleic acids that could not be detected by usual spectroscopic methods. PMID:7506105

  1. Resolving Acetylated and Phosphorylated Proteins by Neutral Urea Triton-Polyacrylamide Gel Electrophoresis, NUT-PAGE

    PubMed Central

    Buehl, Christopher J.; Deng, Xiexiong; Liu, Mengyu; Hovde, Stacy; Xu, Xinjing; Kuo, Min-Hao

    2014-01-01

    Protein acetylation and phosphorylation can be key modifications that regulate both normal and pathological protein functions. Current gel systems used to analyze modified proteins require either expensive reagents or time–consuming second dimension electrophoresis. In this manuscript, we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. This neutral pH urea Triton-polyacrylamide gel electrophoresis system, or NUT-PAGE, separates proteins based on their charge at pH 7 and generates discrete bands from each acetylated and phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents, and requires only a single dimension of electrophoresis. We are able to demonstrate the effectiveness of this system by analyzing phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative to resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge. Method Summary Here we present a single-dimension neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system affording high-resolution separation of acetylated and phosphorylated proteins. PMID:25109292

  2. 'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining.

    PubMed

    Hochstrasser, D F; Merril, C R

    1988-01-01

    The crosslinker diacrylyl-piperazine produces polyacrylamide gels which display improved electrophoretic separation of proteins and better physical strength. It also produces gels with improved detection of proteins by ammoniacal silver staining by reducing the background. This reduced background provided us with an opportunity to investigate residual background staining caused by the catalytic reagents utilized in the polymerization of acrylamide gels. The commonly used catalyst system, tetramethyl-ethylenediamine and ammonium persulfate was shown to be responsible for the yellow staining background found after a prolonged development time with silver staining. An alternate catalyst system has been designed to decrease further the formation of this background staining. Dimethyl-piperazine or tetramethylethylenediamine, potassium or ammonium persulfate, and sodium thiosulfate are shown to provide for gels which have excellent mechanical and staining characteristics. These catalytic systems produce little background staining despite prolonged development time with the ammoniacal silver stain, and they reduce background staining with the dichromate silver stain. PMID:2484987

  3. Characterization of the proteasome using native gel electrophoresis.

    PubMed

    Elsasser, Suzanne; Schmidt, Marion; Finley, Daniel

    2005-01-01

    Several features of the proteasome make it an excellent subject for analysis by native gel electrophoresis: its size, the multiplicity of variant complexes having proteasome activity, the ease of in-gel assays for proteasome activity, and even its relatively high cellular abundance. Accordingly, native gels have been used to analyze the composition, assembly, gating activity, and binding characteristics of the proteasome. This chapter describes methods for preparing, running, and developing native gels and the proteasome species that are routinely visualized. Additionally, the use of native gels to resolve proteasome complexes present in lysate and to characterize proteasome ligands are described. Following native gel electrophoresis, secondary analyses can be performed, such as activating the core particle, making specific activity assessments, Western blotting of the native gel, resolving native complexes with subsequent SDS-PAGE, and protein identification by mass spectrometry. PMID:16275342

  4. Application of optical methods for dose evaluation in normoxic polyacrylamide gels irradiated at two different geometries

    NASA Astrophysics Data System (ADS)

    Adliene, D.; Jakstas, K.; Vaiciunaite, N.

    2014-03-01

    Normoxic gels are frequently used in clinical praxis for dose assessment or 3-D dose imaging in radiotherapy due to their relative simple manufacturing process under normal atmospheric conditions, spatial stability and well expressed modification feature of physical properties which is related to radiation induced polymerization of gels. In this work we have investigated radiation induced modification of the optical properties of home prepared normoxic polyacrylamide gels (nPAG) in relation to polymerization processes that occur in irradiated gels. Two irradiation geometries were used for irradiation of gel samples: broad beam irradiation geometry of teletherapy unit ROKUS-M with a 60Co source and point source irradiation geometry using 192Ir source of high dose rate afterloading brachytherapy unit MicroSelectron v2 which was inserted into gel via 6 Fr (2 mm thick) catheter. Verification of optical methods: UV-VIS spectrometry, spectrophotometry, Raman spectroscopy for dose assessment in irradiated gels has been performed. Aspects of their application for dose evaluation in gels irradiated using different geometries are discussed. Simple pixel-dose based photometry method also has been proposed and evaluated as a potential method for dose evaluation in catheter based interstitial high dose rate brachytherapy.

  5. Photopolymerized diffusion-defined polyacrylamide gradient gels for on-chip protein sizing.

    PubMed

    Lo, Catherine T; Throckmorton, Daniel J; Singh, Anup K; Herr, Amy E

    2008-08-01

    We report on a facile diffusion-based photopatterning technique for generating linear and non-linear decreasing pore-size gradients in cross-linked polyacrylamide gels. Diffusion of low viscosity polymer precursor solutions and a two-step photopatterning process were used to define the decreasing pore-size gradient gels in a microfluidic format, thus eliminating the need for controlled mixing and delivery of polymer precursor solutions. We present an analytical model of the non-steady state diffusion process and numerically evaluate that model for direct comparison with empirical characterizations of the gradient gels. We show that the analytical model provides an effective means to predict the steepness and linearity of a desired gradient gel prior to fabrication. To assess electrophoretic assay performance in the microfluidic gradient gels, on-chip sizing of protein samples (20-116 kDa) was investigated. Baseline resolution of six proteins was demonstrated in 4 s using 3.5% to 10% polyacrylamide gradient gels. The demonstrated ability to conduct efficient protein sizing in ultra-short separation lengths (0.3 cm) means low applied electric potentials are needed to achieve the electric field strengths required for protein separations. The low required electric potentials relax operating constraints on electrical components, as is especially important for translation of the assay into pre-clinical and clinical settings. The gradient gel fabrication method reported is amenable to adaptation to non-sizing protein assays, as well as integration with upstream sample preparation steps and subsequent orthogonal downstream assays. PMID:18651068

  6. Turning a PAGE: the overnight sensation of SDS-polyacrylamide gel electrophoresis.

    PubMed

    Pederson, Thoru

    2008-04-01

    The zonal separation of proteins on the basis of net charge was initially conducted on paper, then in columns of sucrose and later in gels of starch and polyacrylamide, with appropriate electric fields. Then, in 1964, a graduate student at MIT discovered the power of sodium dodecyl sulfate (SDS) to dissociate the envelope proteins of Escherichia coli and to dramatically enhance their electrophoretic resolution when the detergent was included in the gel. While this Ph.D. thesis work continued, a group at the Albert Einstein College of Medicine published in 1965 the use of SDS to disrupt poliovirus particles and to resolve the proteins in gels containing SDS. This group soon followed with a publication (1966) on the application of this new method to the study of immunoglobulin heavy and light chain synthesis. Because of concurrent advances in gel filtration and other methods of protein separation, SDS gel electrophoresis had its greatest impact not in biochemistry but in cell biology and virology. Ingenious devices were soon introduced that facilitated the application of this method to radioactive protein mixtures, followed by the introduction of slab gels for the simultaneous resolution of multiple samples in parallel lanes in a single run. As we today routinely perform "SDS PAGE" (as the method become known, to the great irritation of journal copyeditors and nomenclature committees at the time), it is fitting to pause--four decades later, and remember the pioneers who made SDS gel electrophoresis a reality, a true milestone that caught on almost overnight. PMID:18378803

  7. Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis.

    PubMed

    Liu, Jikun; Yang, Shuang; Lee, Cheng S; DeVoe, Don L

    2008-06-01

    In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%. PMID:18449857

  8. High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis1

    PubMed Central

    Laremore, Tatiana N.; Ly, Mellisa; Solakyildirim, Kemal; Zagorevski, Dmitri V.; Linhardt, Robert J.

    2010-01-01

    Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6 hours using continuous-elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong-anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies. PMID:20211145

  9. Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels.

    PubMed Central

    Ferguson, D A; Lambe, D W

    1984-01-01

    Soluble protein extracts of 37 catalase-positive strains of Campylobacter species were examined by polyacrylamide slab gel electrophoresis (PAGE). Electrophoretic banding patterns showed good correlation with biochemical tests and with available DNA homology data in distinguishing species of Campylobacter but did not differentiate subspecies or biotypes. PAGE patterns indicated that Campylobacter coli is a distinct species. Furthermore, the PAGE patterns indicated that C. jejuni and nalidixic acid-resistant thermophilic Campylobacter species (C. laridis) are each distinct species. The protein banding patterns of C. fetus subsp. venerealis and C. fetus subsp. fetus strains were distinctly different from those of the three thermophilic species. Images PMID:6490829

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

    SciTech Connect

    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 results from a commercial densitometer.

  11. Rapid detection of proteins in polyacrylamide electrophoresis gels with Direct Red 81 and Amido Black.

    PubMed

    Choveaux, David; Krause, Robert G E; Goldring, J P Dean

    2012-01-01

    Proteins separated by SDS-polyacrylamide gel electrophoresis need to be stained with organic dyes to be visualized and to enable comparisons to be made between the intensity of protein bands to observe and determine differences in protein concentration. The standard protein staining is with Coomassie Blue R-250. Coomassie staining takes 1 h to complete. Direct Red 81 and Amido Black stain proteins within 10 min. This chapter describes Direct Red 81 and Amido Black staining in comparison to staining with Coomassie Blue R-250. PMID:22585524

  12. A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels

    PubMed Central

    Tang, Xin; Ali, M. Yakut; Saif, M. Taher A.

    2012-01-01

    Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. Traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. Fibronectin (FN), Laminin (LN) and Collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). Pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers strong enough adhesion with ECM proteins such that a pattern can be printed, but weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5–400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces. PMID:23002394

  13. Identification of different quaternary structures of beef heart cytochrome-c oxidase by two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Heinrichs, M; Schnert, H

    1987-11-01

    A two-dimensional gel electrophoresis is described to identify different quaternary structures of the heart cytochrome-c oxidase. Bovine enzyme was purified and separated by discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions in the 1st dimension into several discrete complexes and thereupon shown to be heterodisperse in Triton X-100 and dodecyl maltoside. A discontinuous SDS-polyacrylamide gel electrophoresis in the 2nd dimension was used to determine the subunit composition of the isolated complexes. One of these represents the intact enzyme with 12 different polypeptides while the others have an incomplete subunit composition. PMID:2822485

  14. Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass.

    PubMed

    Cubillos-Rojas, Monica; Amair-Pinedo, Fabiola; Tato, Irantzu; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2012-01-01

    Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and that it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis. PMID:22585488

  15. Classification of Clostridium butyricum based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis.

    PubMed

    Yokota, K; Fujinaga, Y; Inoue, K; Shimazaki, S; Seo, G; Takeshi, K; Nagamachi, E; Oguma, K

    1998-08-01

    Eleven strains of Clostridium butyricum collected from different sources were analysed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and pulsed-field gel electrophoresis (PFGE). The strains could be classified into four groups based on their banding profiles of the proteins extracted from the cells on SDS-PAGE. Group I consisted of seven strains, and these strains were further divided into five subgroups by PFGE. The strains belonging to groups II, III, and IV on SDS-PAGE were also classified into the same II to IV groups by PFGE. These data indicate that grouping of the strains of C. butyricum can be performed by employing both SDS-PAGE and PFGE. PMID:16887639

  16. Characterisation of soil-bound residue fractions of the fungicide dithianon by gel permeation chromatography and polyacrylamide gel electrophoresis.

    PubMed

    Wanner, U; Burauel, P; Führ, F

    2000-04-01

    The degradation of the (14)C-labelled fungicide dithianon in an orthic luvisol was investigated under standardized conditions in comparison to stimulated microbial activity by an amendment of maize straw. The compound is characterized by mineralization losses of approximately 33% and the formation of non-extractable bound residues of approximately 63% in 64 days. Despite the major role of microorganisms in mineralizing this compound, the formation of bound residues is not biotically induced. Gel permeation chromatography and polyacrylamide gel electrophoresis, as different size separation techniques of the humic acids fractions, showed differences in the distribution patterns of non-extractable residues depending on the addition of straw material. The results presented support the existence of humic substances in soil as a micellar system rather than as a biopolymer. PMID:15092966

  17. Entamoeba histolytica: analysis of the trophozoite proteome by two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Leitsch, David; Radauer, Christian; Paschinger, Katharina; Wilson, Iain B H; Breiteneder, Heimo; Scheiner, Otto; Duchêne, Michael

    2005-07-01

    The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing. PMID:15955311

  18. Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol.

    PubMed

    Alonso, A; Visedo, G; Sancho, M; Fernández-Piqueras, J

    1990-04-01

    A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented. PMID:2340828

  19. Simplified sizing of low-density lipoprotein using polyacrylamide gradient gel electrophoresis of plasma.

    PubMed

    Westhuyzen, J; Graham, S D; Rasiah, R L; Saltissi, D

    1997-01-01

    Low-density lipoprotein (LDL) particles can be separated into subfractions according to size by non-denaturing polyacrylamide gradient gel electrophoresis. Established research methods require specialised equipment and are frequently unsuited to the clinical laboratory. In this study, we utilised a colour flat bed scanner in conjunction with shareware image analysis software to compare LDL particle diameters of isolated LDL with LDL in whole plasma. LDL was isolated by ultracentrifugation and electrophoresed on 3-13% gels (Gradipore; Sydney, Australia) for 2400 Volt-hours in parallel with plasma and molecular size standards. Coomassie Blue-stained gels were scanned in reflexive mode using a colour flat-bed scanner and Adobe Photoshop 3.0 software. Density traces of each lane were obtained using NIH Image software (public domain, USA). LDL particle diameters were determined from calibration curves of the log of molecular diameter of standards against migration distance. There was a good correlation between LDL particle diameters obtained using isolated LDL and whole plasma (r = 0.87, P < 0.001; n = 22). However, the group means (+/- S.D.) (24.7 +/- 0.6 and 24.8 +/- 0.5 nm respectively) were statistically different on the paired t-test (P < 0.05). It is unclear whether this numerically small difference is due to alterations in LDL during the longer preparative procedures for LDL, or to matrix effects during electrophoresis of plasma samples. In conclusion, plasma samples stained with Coomassie Blue and scanned with a colour flat bed scanner can conveniently be used for LDL particle sizing by non-denaturing polyacrylamide gradient gel electrophoresis. PMID:9156560

  20. Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.

    PubMed

    Ladner, Carol L; Edwards, Robert A; Schriemer, David C; Turner, Raymond J

    2006-04-01

    Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels. PMID:16579625

  1. A rapid and effective method for silver staining of PCR products separated in polyacrylamide gels.

    PubMed

    Liang, Qingzhi; Wen, Dingqing; Xie, Jianghui; Liu, Liqin; Wei, Yongzan; Wang, Yicheng; Shi, Shengyou

    2014-09-01

    With the development of molecular quantitative genetics, particularly, genetic linkage map construction, quantitative trait loci mapping or genes fine mapping and association analysis etc., more and more PCR products separated in polyacrylamide gels need to be silver-stained. However, conventional silver-staining procedures are complicated and time-consuming as they require a lot of preparation and handling of several solutions prior to use. In this study, a simple and rapid protocol for silver staining of PCR products was developed. The number of steps was reduced compared to conventional protocols, thus achieving detection of PCR products in 7 min, saving time and resources. Fixation and staining solution and developing solution in present staining procedure allowed a reutilization for 12 and 8 times, respectively, reducing the cost greatly. Meanwhile, the sensitivity was significantly improved with the improved method and the minimum of 0.097 ng/μL of DNA amount can be detected in denaturing polyacrylamide gel. The protocol developed in this study will facilitate the development of molecular quantitative genetics. PMID:24789566

  2. Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing.

    PubMed

    Herr, Amy E; Singh, Anup K

    2004-08-15

    A new method for on-chip sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins is reported. Miniaturization of SDS-PAGE has attracted significant attention because it offers rapid analysis times, excellent resolution, high throughput, and the potential for integration and automation, as compared to conventional counterparts. The presented on-chip SDS-PAGE technique employed photolithographically patterned, cross-linked gels fabricated in situ in <20 min. The effects of sieving gel composition on the migration properties of fluorescently labeled protein standards (ranging in molecular weight from 14.2 to 66 kDa) were quantified, as was the ability of the gels to function as a sieving matrix for biologically relevant species. Ferguson analysis was employed to calculate retardation coefficients and free solution mobilities. In conjunction with fluorescence imaging, the on-chip SDS-PAGE separation mechanism was evaluated in terms of separation performance indexes, as well as limiting behaviors (i.e., free solution mobility, exclusion characteristics). The photolithographically fabricated gels employed for on-chip SDS-PAGE allowed rapid (<30 s) separations of proteins in short separation lengths (4 mm) with efficiencies as high as 4.41 x 10(5) plates/m. The on-chip SDS-PAGE separations were approximately 100 times faster than conventional slab gel SDS-PAGE (60 min) and occurred in a fraction of the separation length required by slab gels. The migration behavior of protein standards correlated well with molecular weight and allowed molecular weight determination for interleukin-2, fibroblast growth factor, insulin-like growth factor, and tetanus toxin C-fragment. PMID:15307783

  3. Model and field studies of the degradation of cross-linked polyacrylamide gels used during the revegetation of slate waste.

    PubMed

    Holliman, Peter J; Clark, Jennifer A; Williamson, Julie C; Jones, Davey L

    2005-01-01

    Cross-linked polyacrylamide gels are increasingly being used in environmental restoration schemes and horticulture as a means of enhancing water supply to plants. However, the environmental impact of cross-linked polyacrylamide gel deployment in soil remains poorly understood. This study assessed the chemical, physical and biological properties of new and field-conditioned cross-linked polyacrylamide gels. Both monomeric acrylamide (11 microg l(-1)) and acrylic acid (285 microg l(-1)) were observed in new gel; however, the levels of monomers in field-conditioned gels (1-6 years old) were very low (acrylamide <1 microg l(-1); acrylic acid <7 microg l(-1)). Generally, freeze-thaw processes and exposure to UV radiation had little effect on gel acrylic acid and acrylamide concentrations. However, elevated temperatures (35 degrees C) caused a significant release of up to 144 mug l(-1) of acrylamide and 453 microg l(-1) of acrylic acid in new gel and up to 25 microg l(-1) of acrylamide and 157 microg l(-1) of acrylic acid in field-conditioned gels. In contrast, gel water holding capacity was highly dependent upon environmental conditions (UV exposure and freeze/thaw cycles produced the greatest loss of water holding in new gels) and gel age. Optical microscopy revealed that after placement in the field the gels became increasingly colonised over time by fungi and bacteria. In enrichment cultures, we were unable, however, to demonstrate microbial growth when cross-linked polyacrylamide was used as the sole nitrogen source. In summary, under a range of conditions cross-linked polyacrylamide did not release acrylamide above legally permitted limits, with the exception of gel subjected to elevated temperatures. However, their capacity for holding water decreased sharply within 18 months. We therefore conclude that cross-linked polyacrylamide placed in soil is relatively stable with respect to the production of potentially toxic acrylamide, a species with a short half-life, which degrades to the much less toxic acrylic acid. However, the loss of water holding capacity raises questions about its long-term effectiveness in land restoration schemes as this is the main reason it is used in this role. PMID:15589246

  4. Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.

    PubMed

    Steinberg, T H; Pretty On Top, K; Berggren, K N; Kemper, C; Jones, L; Diwu, Z; Haugland, R P; Patton, W F

    2001-07-01

    The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes. PMID:11503209

  5. Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.

    PubMed

    Vézina, C A; Milne, R W; Weech, P K; Marcel, Y L

    1988-05-01

    The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3411236

  6. Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    Otani, Mieko; Taniguchi, Taizo; Sakai, Akiko; Seta, Jouji; Kadoyama, Keiichi; Nakamura-Hirota, Tooru; Matsuyama, Shogo; Sano, Keiji; Takano, Masaoki

    2011-07-01

    We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4-5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS. PMID:21384102

  7. Centrifuge-blotting of proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    PubMed

    Hermansen, L F; Pedersen, O; Sletten, K

    1993-12-01

    A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifuge-blotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)-polyacrylamide gel with 1 M KCl, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut-off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N-terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%-30% has been obtained. PMID:8137793

  8. Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

    PubMed

    Ni, Mao-Wei; Ye, Wei-Jian; Cong, Wei-Tao; Hong, Guo-Ying; Zhu, Zhong-Xin; Duan, Yuan-Meng; Zhou, Xuan; Jin, Li-Tai

    2013-12-01

    As a non-covalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence-based dye for detecting proteins both in 1-D and 2-D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which similars to that of glutaraldehyde (GA)-silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces. PMID:24105885

  9. Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates.

    PubMed

    Fulgentini, Lorenzo; Marangoni, Roberto; Colombetti, Giuliano

    2008-06-01

    An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels. PMID:18548458

  10. 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

  11. Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis.

    PubMed

    Li, Fuhou; Liang, Jingdan; Wang, Weixia; Zhou, Xiufen; Deng, Zixin; Wang, Zhijun

    2014-11-15

    Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-resolution clear native/native PAGE, and native/SDS-PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS-PAGE is much higher than that of blue native/SDS-PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor. PMID:25150108

  12. Simplified method for the diameter sizing of serum low-density lipoprotein using polyacrylamide gradient gel electrophoresis.

    PubMed

    Tsukamoto, Hideko; Takei, Izumi; Ishii, Keiko; Watanabe, Kiyoaki

    2004-01-01

    The appearance of small, dense, low-density lipoprotein in serum has been demonstrated to be associated with increased risk of coronary artery disease. The molecular diameter of low-density lipoprotein is usually measured on the basis of mobility differences on polyacrylamide gel electrophoresis. However, since mobility assessed by this method is seriously affected by the increased levels of serum free fatty acids associated with hypertriglyceridemia, we used polyacrylamide gradient gel electrophoresis to eliminate the interference by fatty acids and devised a simple, precise method of polyacrylamide gradient gel electrophoresis to measure the diameter of small, dense, low-density lipoproteins in serum. We used apoferritin and thyroglobulin, which have a molecular diameter of 12.2 nm and 17.0 nm, respectively, and standard low-density lipoprotein particles having a diameter of 25.7 and 27.0 nm as calibrators, estimated by measurement of negative staining of electron microscopy. We also included apoferritin as an internal standard for polyacrylamide gradient gel electrophoresis. The only stain used was Coomassie brilliant blue, and it was used for lipoprotein staining. When we used low-density lipoprotein of 25.73 nm in diameter as a quality control specimen, the coefficient of variation of the size measurements obtained by our method was less than 1.2%. The new method markedly improved the laboratory procedure for measuring the diameter of low-density lipoproteins. PMID:15497465

  13. Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis

    SciTech Connect

    Takai, N.A.; Filetti, S.; Rapoport, B.

    1981-01-01

    Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of /sup 125/I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.

  14. Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA.

    PubMed

    Suyama, Y

    1986-01-01

    Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNA(leu), 2 for tRNA(met), and 1 each for tRNA(phe), tRNA(trp) and tRNA(tyr). The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytosolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a non-treated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. 3H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs. PMID:3127061

  15. Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels.

    PubMed

    Hatch, Anson V; Herr, Amy E; Throckmorton, Daniel J; Brennan, James S; Singh, Anup K

    2006-07-15

    The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics. PMID:16841920

  16. Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels.

    PubMed

    Sneve, Marianne Lunde; Øverbye, Anders; Fengsrud, Monica; Seglen, Per O

    2005-01-01

    Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles. PMID:16874067

  17. Mechanosensitivity of astrocytes on optimized polyacrylamide gels analyzed by quantitative morphometry

    NASA Astrophysics Data System (ADS)

    Moshayedi, Pouria; Costa, Luciano da F.; Christ, Andreas; Lacour, Stephanie P.; Fawcett, James; Guck, Jochen; Franze, Kristian

    2010-05-01

    Cells are able to detect and respond to mechanical cues from their environment. Previous studies have investigated this mechanosensitivity on various cell types, including neural cells such as astrocytes. In this study, we have carefully optimized polyacrylamide gels, commonly used as compliant growth substrates, considering their homogeneity in surface topography, mechanical properties, and coating density, and identified several potential pitfalls for the purpose of mechanosensitivity studies. The resulting astrocyte response to growth on substrates with shear storage moduli of G' = 100 Pa and G' = 10 kPa was then evaluated as a function of coating density of poly-D-lysine using quantitative morphometric analysis. Astrocytes cultured on stiff substrates showed significantly increased perimeter, area, diameter, elongation, number of extremities and overall complexity if compared to those cultured on compliant substrates. A statistically significant difference in the overall morphological score was confirmed with an artificial intelligence-based shape analysis. The dependence of the cells' morphology on PDL coating density seemed to be weak compared to the effect of the substrate stiffness and was slightly biphasic, with a maximum at 10-100 µg ml - 1 PDL concentration. Our finding suggests that the compliance of the surrounding tissue in vivo may influence astrocyte morphology and behavior.

  18. A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma.

    PubMed

    Pérusse, M; Pascot, A; Després, J P; Couillard, C; Lamarche, B

    2001-08-01

    Low plasma levels of HDL cholesterol have been associated with an increased risk of coronary heart disease. HDL particles are heterogeneous with respect to size and apolipoprotein content. The objective of the present study was to develop a method to generate lipid-stainable calibrators that would allow the assessment of HDL particle size from whole plasma, using polyacrylamide gradient gel electrophoresis (PAGGE). Lipid-stainable HDL calibrators were obtained by subjecting isolated red blood cells to hemolysis either by freezing at -20 or -80 degrees C overnight or by rapid exposure to liquid nitrogen and mixing of the hemolysis products with plasma aliquots. All three methods were highly reproducible in producing Sudan black lipid-stainable HDL calibrators ranging from 75 to 200 A. The assessment of HDL particle size with these lipid-stainable HDL calibrators was also highly reproducible, with a coefficient of variation below 5.5%. These lipid-stainable HDL calibrators simplify the assessment of HDL particle size by PAGGE using whole plasma, without the need for costly, time-consuming ultracentrifugation procedures. PMID:11483636

  19. [Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis].

    PubMed

    Fine, J M; Lambin, P; Steinbuch, M

    1975-09-01

    Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose. PMID:59941

  20. Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF.

    PubMed

    Hellman, Jukka

    2007-10-01

    Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS. PMID:17722206

  1. High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation.

    PubMed

    Chettibi, S; Lawrence, A

    1989-01-01

    A new and simple gel electrophoretic method is described which enables the protein and polypeptide components of bee venom to be resolved on a single gel. The electrophoretic method allows octapeptides to be resolved and species as small as decapeptides can be detected at high sensitivity using the Coomassie blue staining method without prior fixation. This has been achieved by replacing acetic acid by propionic acid in acid/urea polyacrylamide gels and by controlling the amount of TEMED catalyst for the polymerisation of high concentration gels in order to obtain a low effective pore size. We demonstrated the value of ethanol precipitation as a rapid and efficient desalting the fractionation technique and propose that it could be used in combination with gel filtration to purify many of the peptides to homogeneity. PMID:2781578

  2. Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.

    PubMed

    Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

    1996-08-01

    The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

  3. Immobilization of microbial cells in crosslinked, prepolymerized, linear polyacrylamide gels: antibiotic production by immobilized Streptomyces clavuligerus cells

    SciTech Connect

    Freeman, A.; Aharonowitz, Y.

    1981-12-01

    A mild method for the immobilization of whole microbial cells has been developed. Cells were suspended in a solution of preformed, linear, water-soluble polyacrylamide chains, partially substituted with acylhydrazide groups. The prepolymerized backbone polymer was crosslinked, in the presence of viable cells, by stoichiometric amounts of dialdehydes such as glyoxal, glutardialdehyde, and periodate-oxidized polyvinyl alcohol. The crosslinking reaction, carried out in cold, neutral physiological conditions resulted in cells entrapped in gels with physical properties similar to those of the common polyacrylamide gels. However, cell damage generally caused by the acrylamide monomer was avoided. Resting Streptomyces clavuligerus cells, possessing a high capacity for antibiotic production, were entrapped according to this procedure. These immobilized cells produced cephalosporins continuously for 96 hours with yields similar to those of free resting cells. The same cells, when immobilized by direct polymerization of acrylamide monomers, yielded significantly lower amounts of antibiotics. (Refs. 19).

  4. Fiber based optical tweezers for simultaneous in situ force exertion and measurements in a 3D polyacrylamide gel compartment.

    PubMed

    Ti, Chaoyang; Thomas, Gawain M; Ren, Yundong; Zhang, Rui; Wen, Qi; Liu, Yuxiang

    2015-07-01

    Optical tweezers play an important role in biological applications. However, it is difficult for traditional optical tweezers based on objective lenses to work in a three-dimensional (3D) solid far away from the substrate. In this work, we develop a fiber based optical trapping system, namely inclined dual fiber optical tweezers, that can simultaneously apply and measure forces both in water and in a 3D polyacrylamide gel matrix. In addition, we demonstrate in situ, non-invasive characterization of local mechanical properties of polyacrylamide gel by measurements on an embedded bead. The fiber optical tweezers measurements agree well with those of atomic force microscopy (AFM). The inclined dual fiber optical tweezers provide a promising and versatile tool for cell mechanics study in 3D environments. PMID:26203364

  5. Fiber based optical tweezers for simultaneous in situ force exertion and measurements in a 3D polyacrylamide gel compartment

    PubMed Central

    Ti, Chaoyang; Thomas, Gawain M; Ren, Yundong; Zhang, Rui; Wen, Qi; Liu, Yuxiang

    2015-01-01

    Optical tweezers play an important role in biological applications. However, it is difficult for traditional optical tweezers based on objective lenses to work in a three-dimensional (3D) solid far away from the substrate. In this work, we develop a fiber based optical trapping system, namely inclined dual fiber optical tweezers, that can simultaneously apply and measure forces both in water and in a 3D polyacrylamide gel matrix. In addition, we demonstrate in situ, non-invasive characterization of local mechanical properties of polyacrylamide gel by measurements on an embedded bead. The fiber optical tweezers measurements agree well with those of atomic force microscopy (AFM). The inclined dual fiber optical tweezers provide a promising and versatile tool for cell mechanics study in 3D environments. PMID:26203364

  6. Proteins specified by herpes simplex virus. Staining and radiolabeling properties of B capsid and virion proteins in polyacrylamide gels.

    PubMed

    Gibson, W; Roizman, B

    1974-01-01

    ANALYSES OF THE STRUCTURAL PROTEINS OF HERPES SIMPLEX VIRIONS AND OF CAPSIDS CONTAINING VIRAL DNA (B CAPSIDS), AFTER ELECTROPHORESIS IN POLYACRYLAMIDE GELS, REVEALED CONSIDERABLE VARIABILITY IN THEIR PROPERTIES WITH RESPECT TO: (i) retention of Coomassie brilliant blue (CBB) and fast green stains during destaining, (ii) relative optical absorbance of the CBB-protein complex at different wavelengths, (iii) relative efficiency with which (14)C-amino acids are incorporated during early and late periods of the infection cycle, and (iv) capacity to be phosphorylated in vivo. In addition, it was found that protein 22a of B capsids, which does not have an electrophoretically identical counterpart in virions, shares a relatively unique set of staining and radiolabeling properties with virion protein 22, which has a slightly more rapid electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. PMID:4129836

  7. 2-D gel densitometer for high-contrast and selective imaging of chlorophyll-containing protein complexes separated by non-denaturing polyacrylamide gel electrophoresis.

    PubMed

    Ilík, Petr; Krchnák, Pavel; Tomek, Pavel; Naus, Jan

    2002-05-31

    In this work, we present a home-made two-dimensional (2-D) CCD imaging system for the monochromatic densitometry of plane gels and its application to the imaging and densitometry of chlorophyll (Chl)-containing proteins separated by non-denaturing polyacrylamide gel electrophoresis. The monochromatic imaging of separated green bands at the wavelengths corresponding to their absorption band increases their contrast. This allows a better visualization of the faint-green bands in the gel and using of samples with lower Chl content for the electrophoresis. By the comparison of 2-D densitograms of the same gel illuminated with 670 and 650 nm lights, that is, at the red absorption maximum of Chl a and b, respectively, we achieved a selective imaging of the complexes with different Chl a/b ratio. This approach was used to specify an unknown band that appeared in the gel of the sample prepared from the thylakoid membranes of preheated barley leaves. PMID:12088887

  8. Detection of antimicrobial (poly)peptides with Acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

    PubMed

    Porter, Edith; Valore, Erika V; Anouseyan, Rabin; Salzman, Nita H

    2015-01-01

    Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1. PMID:25253251

  9. Simple and cost effective apparatus for silver staining of polyacrylamide gels with sequential reagents addition and real time monitoring.

    PubMed

    Maurye, Praveen; Basu, Arpita; Gupta, Angshuman

    2014-06-01

    Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals. PMID:24388443

  10. Multifunctional graphene incorporated polyacrylamide conducting gel electrolytes for efficient quasi-solid-state quantum dot-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Duan, Jialong; Tang, Qunwei; Li, Ru; He, Benlin; Yu, Liangmin; Yang, Peizhi

    2015-06-01

    Pursuit of a high efficiency and stability has been a persistent objective for quantum dot-sensitized solar cells (QDSCs). Here we launch a strategy of synthesizing graphene implanted polyacrylamide (PAAm-G) conducting gel electrolytes for quasi-solid-state QDSCs. With an aim of elevating the dosage of S2-/Sx2- redox couples and therefore charge-transfer ability, both osmotic press across the PAAm-G and capillary force within the three-dimensional micropores are utilized as driving forces. A promising power conversion efficiency of 2.34% is recorded for the QDSCs by optimizing graphene dosage in the conducting gel electrolyte. The enhanced conversion efficiency of solar cell is attributed to the expanded catalytic area from counter electrolyte/electrolyte interface to both interface and the conducting gel electrolyte.

  11. Immunochemical characterization of Mycobacterium leprae antigens by the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP) using patients' sera.

    PubMed Central

    Klatser, P R; van Rens, M M; Eggelte, T A

    1984-01-01

    In this study the SDS-polyacrylamide gel electrophoresis immunoperoxidase (SGIP) assay was used for characterizing the antigenic components of Mycobacterium leprae using patients' sera. This technique involved the separation of mycobacterial sonicates on SDS-polyacrylamide gels, longitudinal sectioning of the gels, incubation with patients' sera and visualization of the antigen-antibody complexes by the indirect immunoperoxidase technique. A number of antigens present in M. leprae sonicates were recognized by leprosy patients' sera, some of which were seen in other mycobacteria as well. Antibody binding to a 33 kD antigen, present in both M. leprae and BCG sonicates, was reduced only in the latter after 6 months of multiple drug treatment of one patient. It is suggested that this is a common mycobacterial antigen with one or more M. leprae specific determinants. Several antigens were identified only in M. leprae sonicates, only by leprosy patients: a 12, 22, 28, 36, 41 and 86 kD component. These antigens lost their antigenicity after trypsin treatment, but were heat stable. Such M. leprae specific antigens may be useful for immunodiagnosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6378452

  12. Identification and mapping of human saphenous vein medial smooth muscle proteins by two-dimensional polyacrylamide gel electrophoresis.

    PubMed

    McGregor, E; Kempster, L; Wait, R; Welson, S Y; Gosling, M; Dunn, M J; Powel, J T

    2001-11-01

    Changing smooth muscle phenotype and abnormal cell proliferation are important features of vascular pathology, including the failure of saphenous vein bypass grafts. We have characterised and mapped protein expression in human saphenous vein medial smooth muscle, using two-dimensional (2-D) polyacrylamide gel electrophoresis. The 2-D system comprised a nonlinear immobilised pH 3-10 gradient in the first dimension (separating proteins with isoelectric point values between pH 3-10), and 12%T total gel concentration sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension (separating proteins in the range 14,000-200,000 Daltons). Using a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and partial amino acid sequencing by nanospray tandem mass spectrometry, a subset of 149 protein spots was analysed, with 129 protein spots being identified and mapped. The data presented here are an important addition to the limited knowledge of venous medial smooth muscle protein expression in vivo. Our protein map will facilitate the identification of proteins differentially expressed in human saphenous vein bypass grafts. In turn, this may lead to the elucidation of molecular events involved in saphenous vein bypass graft failure. The map should also provide a basis for comparative studies of protein expression in vascular smooth muscle of varying origins. PMID:11922600

  13. Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.

    PubMed

    Sobieszek, A

    1994-01-01

    Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described. PMID:7859701

  14. High resolution two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation.

    PubMed

    Marshall, T; Williams, K M

    1988-03-01

    The proteins of cheese are rapidly solubilised by heating to 95 degrees C in buffered 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis or high resolution two-dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink-violet components as detected by Coomassie Blue staining. PMID:2466651

  15. The use of N,N,N',N'-tetramethylphenylenediamine to detect peroxidase activity on polyacrylamide electrophoresis gels.

    PubMed

    Butler, M J; Lachance, M A

    1987-05-01

    N,N,N',N'-Tetramethylphenylenediamine (TMPD) acts as an effective indicator of peroxidase activity on polyacrylamide electrophoresis gels. The test is easy to perform, rapid, sensitive, and reliable. The procedure produces vivid bright blue bands (Wursters blue) on a clear background. TMPD and Wursters blue did not interfere with a number of other electrophoresis stains subsequently applied. These included total protein staining with Coomassie blue, and a number of pigment producing electrophoresis stains used to investigate melanogenesis-related enzymes in the black yeast Phaeococcomyces sp. PMID:2440348

  16. Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. II. Effect of D-glucoside derivatives on development.

    PubMed

    Bozzaro, S; Roseman, S

    1983-11-25

    In an accompanying report (Bozzaro, S., and Roseman, S. (1983) J. Biol. Chem. 258, 13882-13889), evidence is presented that the slime mold Dictyostelium discoideum contains three cell surface receptors specific for D-glucose, D-mannose, and N-acetyl-D-glucosamine, respectively. The synthetic probes used for these studies consisted of the sugars covalently linked to polyacrylamide gels. In the present experiments, starved cells were placed on these and other immobilized sugars to determine whether the sugar derivatives influenced normal development in this organism. When D. discoideum cells are on a solid surface under water, they form aggregation centers and strands of cells (which radiate from the center), send "signals" i.e. pulses of cyclic AMP from the center down the strands, and finally, after cells in the strands migrate to the center, form tight aggregates. These results were obtained on all polyacrylamide gel derivatives tested except one class, derivatives of D-glucose (O- and S-glucosides, cellobiosides, and maltosides). On these gels, aggregation centers and strands formed normally, but at a certain point stopped "signaling" and suddenly dissociated, with the cells rapidly migrating away from one another by negative chemotaxis (see Appendix to this report). Furthermore, a simultaneous dissociation of several centers was often observed. Following a brief period of random movement after dissociation, aggregation centers once again formed and the cycle was repeated. This cycle was repeated as often as 30 times or more over a 24-h period. The cells on the glucoside gels became aggregation-competent at the same time as the control cells, and the adhesion-dissociation cycle appeared to have no effect on the synthesis of some developmentally regulated proteins, such as UDP-glucose pyrophosphorylase. Interpretations of the phenomenon and its potential for studying gene regulation in this organism are discussed. PMID:6315710

  17. Mutation analysis of fragile X syndrome by Southern blot, radioactive PCR, silver-stained polyacrylamide gel and DIG DNA

    SciTech Connect

    Lee, Sook-Hwan; Kim, Un-Kyung; Chung-Woong, M.S.

    1994-09-01

    Fragile X syndrome is the most common inherited form of mental retardation. In fragile X syndrome, the underlying mutation is caused by an expansion of the CTG triplet in the 5{prime} untranslated region of the FMR-1 gene located at Xq27.3 and diagnosed by methylation of the associated CpG island. This disorder becomes clinically manifested when the mutation is caused by an expansion of (CGG)n reaching a threshold of about 600bp (200 repeats). The number of inserted repeats increases through the generation. We have analyzed fragile X syndrome by 4 different methods: Southern blot, radioactive PCR, polyacrylamide gel and DIG DNA labeling/detection techniques. Southern blot and DIG DNA labeling/detection by double DNA digestion with EcoRI and EagI reveals both the presence of the mutation and the methylation status. Radioactive PCR and silver-stained polyacrylamide gel is a rapid and sensitive technique to define the unaffected carriers and NTMs, but it is difficult to amplify such a highly GC-rich sequence. Further testing in other fragile X patients is currently in progress.

  18. Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels.

    PubMed

    Snabes, M C; Boyd, A E; Bryan, J

    1981-09-01

    Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues. PMID:6793603

  19. Electrophoretic transfer from polyacrylamide gel to nitrocellulose sheets, a new method to characterize multilocus enzyme genotypes of Klebsiella strains.

    PubMed

    Combe, M L; Pons, J L; Sesboue, R; Martin, J P

    1994-01-01

    A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-mul samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains. PMID:16349155

  20. The Use of Polyacrylamide Gels for Mechanical Calibration of Cartilage – A Combined Nanoindentation and Unconfined Compression Study

    PubMed Central

    Li, Cheng; Allen, Jessica; Alliston, Tamara; Pruitt, Lisa A.

    2015-01-01

    This study investigates polyacrylamide (PA) gel as a calibration material to measure the nanomechanical compressive modulus of cartilage using nanoindentation. Both nanoindentation and unconfined compression testing were performed on PA gel and porcine rib cartilage. The equilibrium moduli measured by the two methods were discernable. Nanoindentation has the advantage of distinguishing between spatially dependent constituent properties that affect tissue mechanical function in heterogeneous and hierarchically structured tissues such as cartilage. Both sets of measurements exhibited similar positive correlation with increasing gel crosslinker concentration. The compressive modulus measurements from compression in the PA gels ranged from 300 kPa—1.4 MPa, whereas those from nanoindentation ranged from 100 kPa—1.1MPa. Using this data, a method for relating nanoindentation measurements to conventional mechanical property measurements is presented for porcine rib cartilage. It is shown that based on this relationship, the local tissue modulus as measured from nanoindentation (1.1—1.4 MPa) was able to predict the overall global modulus of the same sample of rib cartilage (2.2 MPa), as confirmed by experimental measurements from unconfined compression. This study supports the use of nanoindentation for the local characterization of cartilage tissues and may be applied to other soft tissues and constructs. PMID:21783163

  1. Improvements in allelic discrimination of microsatellite markers using denaturing polyacrylamide gel electrophoresis.

    PubMed

    Symons, R C; Marshall, V M; Foote, S J

    2000-08-01

    Poor resolution, retarded progress of DNA through gels, and variable sizing of DNA fragments between and within gels hinder accurate genotyping of some simple sequence length polymorphism (SSLP) markers with the Perkin Elmer Applied Biosystems 377 Sequenator. These problems are similar to renaturation related problems observed in DNA sequencing gels. PCR products especially susceptible to these problems are shown to have higher melting temperatures (T(m)) than others. Gels containing increased concentrations of denaturants allow greater accuracy in allelic discrimination. This is especially beneficial where quantification is necessary. PMID:10920238

  2. Highly sensitive detection of ATPase activity in native gels.

    PubMed

    Suhai, Tina; Heidrich, Nicolas G; Dencher, Norbert A; Seelert, Holger

    2009-10-01

    Native electrophoresis is a powerful tool for the separation of intact protein complexes. By incubating such gels in a suitable reaction solution, specific enzyme activities can be screened comprehensively. The recent standard procedure for determination of ATP hydrolysis activity in blue or clear native gels is based on formation of a lead phosphate precipitate. The resulting white bands are challenging for detection and documentation of low activities. For the analysis of photosynthetic ATP synthases, the method has to be adapted to deregulate the inhibition of latent ATPase functions. Therefore, we introduced an incubation of gels in detergent solution, whereby taurodeoxycholate turned out to be the most efficient activator. In order to detect low ATPase activities, a short additional incubation step subsequent to the formation of lead phosphate is recommended. By adding ammonium sulfide, the white bands are converted into brownish-black bands of lead sulfide. Our new procedure sustains the linear quantitation range of the original lead phosphate protocol and moreover expands the detection limit. PMID:19784950

  3. A novel [Ag(NH3)2]+ probe for chemiluminescent imaging detection of proteins after polyacrylamide gel electrophoresis.

    PubMed

    Xiong, Xin; Wang, Zhenzhen; Baeyens, Willy R G; Delanghe, Joris R; Huang, Zhi; Huang, Guangming; Ouyang, Jin

    2007-08-01

    The development of a novel [Ag(NH3)2]+ probe chemiluminescence (CL)-based imaging method for the detection of various proteins after PAGE is described. The detection is based upon the probe [Ag(NH3)2]+ catalyzing the CL reaction of the luminol-potassium persulfate system. The proposed method detects various proteins labeled by [Ag(NH3)2]+ and expands the application scope to SDS gels. It also detects proteins directly in polyacrylamide gels, without tedious transferring procedures. Furthermore, successful identification of proteins by peptide mass profiling using ionization MS was easily performed, and no pretreatments of gel prior to digestion are needed. Detection limits for standard marker proteins match CBB-R250 staining and the linear dynamic range is superior to CBB-R250 staining and silver staining. The CL imaging conditions, including luminescent reagents, silver ion concentration, the ammonia-controlled system and the washing reagents parameters have also been optimized. PMID:17610207

  4. Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.

    PubMed

    Ehring, H; Strömberg, S; Tjernberg, A; Norén, B

    1997-01-01

    A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases. PMID:9404036

  5. Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

    PubMed

    Vincenzi, Simone; Curioni, Andrea

    2005-01-01

    An anomalous electrophoretic behavior of a chitinase isoform present in both grape (Vitis vinifera L.) berries and wine was observed in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. A progressive shift of the relative molecular mass M(r) of the enzyme (from approximately 30,500 up to approximately 57,700) with increasing glycol chitin concentration in the gels up to 0.1% was revealed when samples were electrophoresed under nonreducing conditions, whereas the presence of glycol chitin had no effects when samples were reduced before SDS-PAGE separation. The M(r) of other grape and wine chitinase isoforms as well as that of the chitinase from pomegranate (Punica granatum L.) fruit was unaffected by the presence of the substrate in the gel under both reducing and nonreducing conditions. Since the enzymes were inactive during the electrophoretic separation, it is likely that the retarding effect of glycol chitin observed specifically for the unreduced chitinase band from grape and wine was due to an interaction between the substrate and a chitin-binding domain different from the catalytic site, such as that typical of class I and class IV chitinases. PMID:15624140

  6. Tryptic peptide analysis of nanogram quantities of proteins: radioiodination of proteins detected by silver staining in polyacrylamide gels

    SciTech Connect

    Arezzo, F.; Rose, K.M.

    1987-12-01

    The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified so that the polypeptides remain suitable for subsequent radioiodination and tryptic peptide analysis. The procedure, which involves a silver-staining/destaining protocol that minimizes crosslinking, is more rapid than many other methods, and can detect as little as 1 ng of protein. Following elimination of silver, the proteins can be radioiodinated and digested with trypsin by a modification of the method described by J. H. Elder, R. A. Pickett, J. Hampton, and R. A. Lerner. Together, these improvements have increased the sensitivity of the tryptic peptide mapping technique by three orders of magnitude and thereby enabled us to perform reproducible structural analysis of femtomolar quantities of proteins.

  7. 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. PMID:26796977

  8. Fluorography--limitations on its use for the quantitative detection of /sup 3/H- and /sup 14/-C-labeled proteins in polyacrylamide gels

    SciTech Connect

    Harding, C.R.; Scott, I.R.

    1983-03-01

    The suitability of fluorography for the detection of /sup 3/H- and /sup 14/C-labeled proteins on polyacrylamide gradient gels has been investigated. If was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.

  9. Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts.

    PubMed

    Hardy, Eugenio; Ramón, José; Saez, Vivian; Báez, Reynier; Tejeda, Yosbel; Ruiz, Amalia

    2008-06-01

    The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha2b) reacted with either a branched-chain (40,000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12,000) and chromatographically purified monoPEG2,40-IFN-alpha2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG2,40 IFN-alpha2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins. PMID:18449861

  10. Demonstration of lipopolysaccharide with O-polysaccharide chains among different heat-stable serotypes of Campylobacter jejuni by silver staining of polyacrylamide gels.

    PubMed Central

    Blake, D C; Russell, R G

    1993-01-01

    Lipopolysaccharide (LPS) from three human and four monkey isolates of Campylobacter jejuni was extracted in high yields that revealed ladder-like structures in polyacrylamide gels by direct silver staining. These observations demonstrate that C. jejuni possesses LPS with O-chain repeating units typical of the family Enterobacteriaceae. The isolates showed differences in the number and electrophoretic mobility of bands in silver-stained gels. Images PMID:7693600

  11. Metachromatic staining patterns of basic proline-rich proteins from rat and human saliva in sodium dodecyl sulfate-polyacrylamide gels.

    PubMed

    Humphreys-Beher, M G; Wells, D J

    1984-01-01

    A series of basic proteins, rich in proline, were isolated from the salivary secretions of humans and rats. These proteins underwent metachromasia after staining with Coomassie brilliant blue R-250 in sodium dodecyl sulfate-polyacrylamide gels. The technique of destaining gels in several changes of 10% acetic acid after a 30-min staining period is a rapid method of general utility for the identification of proline-rich proteins from total cell lysates from other sources besides saliva. PMID:6085627

  12. Normoxic polyacrylamide gel doped with iodine: response versus X-ray energy.

    PubMed

    Gastaldo, Jérôme; Boudou, Caroline; Lamalle, Laurent; Troprès, Irène; Corde, Stéphanie; Sollier, Albéric; Rucka, Günther; Elleaume, Hélène

    2008-12-01

    The basis of Synchrotron Stereotactic Radio-Therapy (SSRT) is the incorporation of high atomic number atoms (iodine, for example) into the tumour mass followed by an irradiation with a monochromatic, low energy, X-ray beam from a synchrotron source. The purpose of the present study was to determine whether polymer gel dosimetry could be used to measure the enhancement of absorbed energy induced by the iodine in the media. We have used a standard nPAG formulation, loaded with NaI and the irradiations were performed either with monochromatic X-rays at the ESRF medical beamline or with a conventional 6 MV X-ray beam from a linear accelerator at the Grenoble University Hospital. We observed sensitivity increase with iodine loaded gels irradiated at low energies, in good agreement with the theoretical iodine dose-enhancement. As expected, the response of the iodine-doped polymer gel was not increased after irradiation with mega-voltage X-rays. We demonstrate in this study that polymer gel dosimeters can be used for measuring dose-enhancement due to iodine presence in SSR treatment. PMID:18602237

  13. Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry.

    PubMed

    Fu, Zujiao; Sun, Yunjun; Xia, Liqiu; Ding, Xuezhi; Mo, Xiangtao; Li, Xiaohui; Huang, Kexue; Zhang, Youming

    2008-07-01

    Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. PMID:18463863

  14. Quantification of AAV particle titers by infrared fluorescence scanning of coomassie-stained sodium dodecyl sulfate-polyacrylamide gels.

    PubMed

    Kohlbrenner, Erik; Henckaerts, Els; Rapti, Kleopatra; Gordon, Ronald E; Linden, R Michael; Hajjar, Roger J; Weber, Thomas

    2012-06-01

    Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity. PMID:22816378

  15. A modified Coomassie blue staining of proteins in polyacrylamide gels with Bismark brown R.

    PubMed

    Choi, J K; Yoon, S H; Hong, H Y; Choi, D K; Yoo, G S

    1996-04-01

    A rapid and sensitive protein staining method employing a mixed dye technique has been developed. Solutions of Coomassie brilliant blue R-250 (CB, 0.2%) and Bismark brown R (BBR, 0.05%) were mixed in the volume ratio of 1:0.75 for staining (final molar ratio, 4.5:1). In this staining, BBR inhibits the binding of CB to gel matrix by forming ion-pairing complex with excess CB; therefore, it reduces staining/destaining times and also enhances the staining effect of CB on protein band. As a result, the mixed dye staining enables complete staining/destaining within 20/25 min and detection of up to 25 ng of bovine serum albumin. PMID:8619499

  16. Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.

    PubMed

    Dyballa, Nadine; Metzger, Sabine

    2009-01-01

    Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches. Several improvements of the original Coomassie protocol(1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution(2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol(3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol(4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins. Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group. PMID:19684561

  17. Polyacrylamide gel 10 years experience: with particular reference to complications from filling of the body of the lip.

    TOXLINE Toxicology Bibliographic Information

    Rish A

    2014-12-01

    BACKGROUND: A review of 242 facial treatments, in 86 different patients, with polyacrylamide gel (PAAG) was carried out by the author between 2003 and 2013.OBJECTIVES: To evaluate by retrospective study the long-term outcomes of PAAG filling for facial contouring. To quantify adverse events and patient comfort in prior mixing 0.3 mL of 2% lidocaine/1 mL PAAG and decanting into a smaller volume syringe.METHOD: Review of clinical records combined with a patient survey.COMPLICATIONS: 11 of 166 (6.6%) lip body infections; 1 of 202 (0.5%) in other sites. Addition of 2% lidocaine (55 treatments/28 patients) reduced lip body infections (8.7% to 5.7%, P < 0.05%) and mean pain score (8/10 to 2/10). No patients sustained any long-term side effects and all (even those infected) were eventually satisfied. Seven lip asymmetries, after infected PAAG drainage, were corrected with further PAAG, showing adverse events to be contamination rather than immune reaction.CONCLUSION: Facial usage of PAAG with lidocaine results in high levels of patient satisfaction and low incidence of side effects (0.5%) except in the lip body (6.6%). Potential users, fearful of complications, may more readily use this cost-effective and long-lasting dermal filler if they avoid injection of the lip body.

  18. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.

    PubMed

    Michaud, D; Cantin, L; Raworth, D A; Vrain, T C

    1996-01-01

    A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans. PMID:8907521

  19. Genetic diversity analysis of faba bean (Vicia faba L.) germplasms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    PubMed

    Hou, W W; Zhang, X J; Shi, J B; Liu, Y J

    2015-01-01

    To investigate genetic diversity and relationships of 101 faba bean (Vicia faba L.), landraces and varieties from different provinces of China and abroad were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A total of 2625 unambiguous and stable bands from 101 germplasms were detected, and 36 different bands were classified according to the electrophoretic mobility patterns of the proteins as determined by the SDS-PAGE analysis, of which 16 were polymorphic. Besides the common bands, the protein bands of 92, 75, 62, 40, 34, 17, and 13 kDa presented the highest frequencies of 92.08, 90.10, 99.01, 95.05, 95.05, 98.02, and 95.05%, respectively. The other 29 polymorphic protein bands showed higher polymorphism with 16.09 polymorphic bands in average. The genetic similarity of the 101 genotypes tested varied from 0.6111 to 0.9722, with an average of 0.7122. Cluster analysis divided the 101 genotypes into six major clusters, which was consistent with the systematic classification of faba bean done in previous studies. The overall results indicated that SDS-PAGE was a useful tool for genetic diversity analysis and laid a solid foundation for future faba bean breeding. PMID:26535710

  20. Detection of rotavirus by a latex agglutination test, Rotalex: comparison with electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay.

    PubMed Central

    Moosai, R B; Alcock, R; Bell, T M; Laidler, F R; Peiris, J S; Wyn-Jones, A P; Madeley, C R

    1985-01-01

    A commercially available latex agglutination test, Rotalex (Orion Diagnostics, Finland), for detecting rotaviruses was evaluated in comparison with four other tests (electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay) routinely used in our laboratories. Although Rotalex was the least complex method, it showed lack of specificity and sensitivity when carried out according to the manufacturer's instructions. Four basic modifications of Rotalex are described. These include the use of Hank's balanced salt solution, increasing the incubation time to 20 min, reading the agglutination result by an experienced observer, and the use of 50 mm square glass plates. The modified procedure gave results which were comparable with those obtained by electron microscopy, immunofluorescence, polyacrylamide gel electrophoresis, and enzyme linked immunosorbent assay. The latter techniques, when used to detect rotavirus, all gave similar results. PMID:2989342

  1. Highly sensitive method for specific, brief, and economical detection of glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the synthesis of a new hydrazide derivative.

    PubMed

    Cong, Weitao; Zhou, Ayi; Liu, Zhiguo; Shen, Jiayi; Zhou, Xuan; Ye, Weijian; Zhu, Zhongxin; Zhu, Xinliang; Lin, Jianjun; Jin, Litai

    2015-02-01

    A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively. PMID:25565298

  2. Comparative two-dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

    PubMed Central

    Ngounou Wetie, Armand G; Wormwood, Kelly L; Charette, Laci; Ryan, Jeanne P; Woods, Alisa G; Darie, Costel C

    2015-01-01

    In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two-dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P-value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC-MS/MS. Alpha-amylase, CREB-binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down-regulated in ASD. Increased expression of proto-oncogene Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin-inducible protein precursor, Mucin-16, Ca binding protein migration inhibitory factor-related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD. PMID:26290361

  3. Comparative two-dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder.

    PubMed

    Ngounou Wetie, Armand G; Wormwood, Kelly L; Charette, Laci; Ryan, Jeanne P; Woods, Alisa G; Darie, Costel C

    2015-11-01

    In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two-dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P-value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC-MS/MS. Alpha-amylase, CREB-binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down-regulated in ASD. Increased expression of proto-oncogene Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin-inducible protein precursor, Mucin-16, Ca binding protein migration inhibitory factor-related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD. PMID:26290361

  4. SU-E-T-105: Development of 3D Dose Verification System for Volumetric Modulated Arc Therapy Using Improved Polyacrylamide-Based Gel Dosimeter

    SciTech Connect

    Ono, K; Fujimoto, S; Akagi, Y; Hirokawa, Y; Hayashi, S; Miyazawa, M

    2014-06-01

    Purpose: The aim of this dosimetric study was to develop 3D dose verification system for volumetric modulated arc therapy (VMAT) using polyacrylamide-based gel (PAGAT) dosimeter improved the sensitivity by magnesium chloride (MgCl{sub 2}). Methods: PAGAT gel containing MgCl{sub 2} as a sensitizer was prepared in this study. Methacrylic-acid-based gel (MAGAT) was also prepared to compare the dosimetric characteristics with PAGAT gel. The cylindrical glass vials (4 cm diameter, 12 cm length) filled with each polymer gel were irradiated with 6 MV photon beam using Novalis Tx linear accelerator (Varian/BrainLAB). The irradiated polymer gel dosimeters were scanned with Signa 1.5 T MRI system (GE), and dose calibration curves were obtained using T{sub 2} relaxation rate (R{sub 2} = 1/T{sub 2}). Dose rate (100-600 MU min{sup −1}) and fractionation (1-8 fractions) were varied. In addition, a cubic acrylic phantom (10 × 10 × 10 cm{sup 3}) filled with improved PAGAT gel inserted into the IMRT phantom (IBA) was irradiated with VMAT (RapidArc). C-shape structure was used for the VMAT planning by the Varian Eclipse treatment planning system (TPS). The dose comparison of TPS and measurements with the polymer gel dosimeter was accomplished by the gamma index analysis, overlaying the dose profiles for a set of data on selected planes using in-house developed software. Results: Dose rate and fractionation dependence of improved PAGAT gel were smaller than MAGAT gel. A high similarity was found by overlaying the dose profiles measured with improved PAGAT gel dosimeter and the TPS dose, and the mean pass rate of the gamma index analysis using 3%/3 mm criteria was achieved 90% on orthogonal planes for VMAT using improved PAGAT gel dosimeter. Conclusion: In-house developed 3D dose verification system using improved polyacrylamide-based gel dosimeter had a potential as an effective tool for VMAT QA.

  5. An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

    PubMed

    Visal-Shah, S; Vrain, T C; Yelle, T C; Nguyen-Quoc, B; Michaud, D

    2001-08-01

    A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology. PMID:11545387

  6. Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes

    SciTech Connect

    Ikeda, S.; Seki, S.; Watanabe, S.; Hatsushika, M.; Tsutsui, K. )

    1991-01-01

    A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-{sup 32}P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.

  7. Three-dimensional polyacrylamide gel-based DNA microarray method effectively identifies UDP-glucuronosyltransferase 1A1 gene polymorphisms for the correct diagnosis of Gilbert's syndrome.

    PubMed

    Song, Jinyun; Sun, Mei; Li, Jiayan; Zhou, Dongrui; Wu, Xuping

    2016-03-01

    Gilbert's syndrome is a mild genetic liver disorder characterized by unconjugated hyperbilirubinemia due to defects in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The T-3279G mutation in the phenobarbital responsive enhancer module (PBREM), the TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of A(TA)6TAA and the G211A mutation in coding exon 1, particularly in Asian populations, of the human UGT1A1 gene are the three common genotypes found in patients with Gilbert's syndrome. Different approaches for detecting the T-3279G, A(TA)6/7TAA and G211A mutations of the UGT1A1 gene have been described. In this study, to the best of our knowledge, we established a three-dimensional polyacrylamide gel-based DNA microarray method for the first time, in order to study UGT1A1 gene polymorphisms. This method, based on a step-by-step three-dimensional polyacrylamide gel-based DNA microarray protocol, successfully identified all possible genotypes of T-3279G, A(TA)6/7TAA and G211A in 20 patients with hyperbilirubinemia. In addition, sequencing was performed to confirm these results. The data from the current study demonstrate that the three-dimensional polyacrylamide gel microarray method has the potential to be applied as a useful, reliable and cost-effective tool to detect the T-3279G, the A(TA)6/7TAA and the G211A mutations of the UGT1A1 gene in patients with hyperbilirubinemia and thereby aid in the diagnosis of Gilbert's syndrome. PMID:26781906

  8. Three-dimensional polyacrylamide gel-based DNA microarray method effectively identifies UDP-glucuronosyltransferase 1A1 gene polymorphisms for the correct diagnosis of Gilbert's syndrome

    PubMed Central

    SONG, JINYUN; SUN, MEI; LI, JIAYAN; ZHOU, DONGRUI; WU, XUPING

    2016-01-01

    Gilbert's syndrome is a mild genetic liver disorder characterized by unconjugated hyperbilirubinemia due to defects in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The T-3279G mutation in the phenobarbital responsive enhancer module (PBREM), the TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of A(TA)6TAA and the G211A mutation in coding exon 1, particularly in Asian populations, of the human UGT1A1 gene are the three common genotypes found in patients with Gilbert's syndrome. Different approaches for detecting the T-3279G, A(TA)6/7TAA and G211A mutations of the UGT1A1 gene have been described. In this study, to the best of our knowledge, we established a three-dimensional polyacrylamide gel-based DNA microarray method for the first time, in order to study UGT1A1 gene polymorphisms. This method, based on a step-by-step three-dimensional polyacrylamide gel-based DNA microarray protocol, successfully identified all possible genotypes of T-3279G, A(TA)6/7TAA and G211A in 20 patients with hyperbilirubinemia. In addition, sequencing was performed to confirm these results. The data from the current study demonstrate that the three-dimensional polyacrylamide gel microarray method has the potential to be applied as a useful, reliable and cost-effective tool to detect the T-3279G, the A(TA)6/7TAA and the G211A mutations of the UGT1A1 gene in patients with hyperbilirubinemia and thereby aid in the diagnosis of Gilbert's syndrome. PMID:26781906

  9. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Protein Banding Patterns among Rhizobium leguminosarum biovar phaseoli Strains Isolated from the Mexican Bean Phaseolus coccineus

    PubMed Central

    Arredondo-Peter, R.; Escamilla, E.

    1993-01-01

    Several rhizobial strains were isolated from Phaseolus coccineus root nodules and were determined to be Rhizobium leguminosarum biovar phaseoli strains after reinfection of the same host plant. These strains were characterized by cultural procedures (growth on different carbon sources and intrinsic antibiotic resistance) and electrophoretic procedures (sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins). Our results showed that these rhizobia are very similar to each other, especially in their electrophoretic protein banding patterns, suggesting that they might belong to isolated populations. Images PMID:16349098

  10. Fluid diversion and sweep improvement with chemical gels in oil recovery processes. [Four types of gels: resorcinol-formaldehyde; colloidal silica; Cr sup 3+ (chloride)-xanthan; and Cr sup 3+ (acetate)-polyacrylamide

    SciTech Connect

    Seright, R.S.; Martin, F.D.

    1992-09-01

    The objectives of this project were to identify the mechanisms by which gel treatments divert fluids in reservoirs and to establish where and how gel treatments are best applied. Several different types of gelants were examined, including polymer-based gelants, a monomer-based gelant, and a colloidal-silica gelant. This research was directed at gel applications in water injection wells, in production wells, and in high-pressure gas floods. The work examined how the flow properties of gels and gelling agents are influenced by permeability, lithology, and wettability. Other goals included determining the proper placement of gelants, the stability of in-place gels, and the types of gels required for the various oil recovery processes and for different scales of reservoir heterogeneity. During this three-year project, a number of theoretical analyses were performed to determine where gel treatments are expected to work best and where they are not expected to be effective. The most important, predictions from these analyses are presented. Undoubtedly, some of these predictions will be controversial. However, they do provide a starting point in establishing guidelines for the selection of field candidates for gel treatments. A logical next step is to seek field data that either confirm or contradict these predictions. The experimental work focused on four types of gels: (1) resorcinol-formaldehyde, (2) colloidal silica, (3) Cr{sup 3+}(chloride)-xanthan, and (4) Cr{sup 3+}(acetate)-polyacrylamide. All experiments were performed at 41{degrees}C.

  11. Mechanism of smectic arrangement of montmorillonite and bentonite clay platelets incorporated in gels of poly(acrylamide) induced by the interaction with cationic surfactants.

    PubMed

    Starodoubtsev, S G; Lavrentyeva, E K; Khokhlov, A R; Allegra, G; Famulari, A; Meille, S V

    2006-01-01

    Structure transitions, induced by the interaction with the cationic surfactant cetylpyridinium chloride in nanocomposite gels of poly(acrylamide) with incorporated suspensions of the two closely related layered clays bentonite and montmorillonite, were studied. Unexpectedly, different behaviors were revealed. X-ray diffraction measurements confirm that, due to the interaction with the surfactant, initially disordered bentonite platelets arrange into highly ordered structures incorporating alternating clay platelets and surfactant bilayers. The formation of these smectic structures also in the cross-linked polymer gels, upon addition of the surfactant, is explained by the existence of preformed, poorly ordered aggregates of the clay platelets in the suspensions before the gel formation. In the case of montmorillonite, smectic ordering of the disordered platelets in the presence of the surfactant is observed only after drying the suspensions and the clay-gel composites. Rheology studies of aqueous suspensions of the two clays, in the absence of both surfactant and gel, evidence a much higher viscosity for bentonite than for montmorillonite, suggesting smaller clay-aggregate size in the latter case. Qualitatively consistent results are obtained from optical micrographs. PMID:16378447

  12. Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer.

    PubMed

    Jin, Li-Tai; Hwang, Sun-Young; Yoo, Gyurng-Soo; Choi, Jung-Kap

    2004-08-01

    A highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver-ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver-dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver-ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver-ion sensitizer. The detection limit of NN-silver stain is 0.05-0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN-silver stain would be useful for routine silver staining of proteins. PMID:15300767

  13. Coloration of silver-stained protein bands in polyacrylamide gels is caused by light scattering from silver grains of characteristic sizes.

    PubMed Central

    Merril, C R; Bisher, M E; Harrington, M; Steven, A C

    1988-01-01

    This study investigates the physical basis of color effects in the detection of proteins in polyacrylamide gels by silver staining. Specifically, the hypothesis that different colors may correlate with the development of silver grains of characteristic sizes was investigated by electron microscopy. Protein bands that stained brown, yellow, and blue were excised from stained gels and prepared for electron microscopy by thin-sectioning. In each case, the size distributions of globular silver grains were determined directly from the electron micrographs. We found that blue bands have larger silver grains (with diameters of 40-100 nm) than yellow (21-39 nm) or brown bands (17-35 nm). On the basis of these and other observations, a general mechanism is proposed whereby chemical specificity of electrophoretically separated proteins is expressed in color-specific silver staining. Images PMID:2448776

  14. Characterization of the aerosol produced by infrared femtosecond laser ablation of polyacrylamide gels for the sensitive inductively coupled plasma mass spectrometry detection of selenoproteins

    NASA Astrophysics Data System (ADS)

    Claverie, Fanny; Pécheyran, Christophe; Mounicou, Sandra; Ballihaut, Guillaume; Fernandez, Beatriz; Alexis, Joël; Lobinski, Ryszard; Donard, Olivier F. X.

    2009-07-01

    A 2D high repetition rate femtosecond laser ablation strategy (2-mm wide lane) previously developed for the detection of selenoproteins in gel electrophoresis by inductively coupled plasma mass spectrometry was found to increase signal sensitivity by a factor of 40 compared to conventional nanosecond ablation (0.12-mm wide lane) [G. Ballihaut, F. Claverie, C. Pécheyran, S. Mounicou, R. Grimaud and R. Lobinski, Sensitive Detection of Selenoproteins in Gel Electrophoresis by High Repetition Rate Femtosecond Laser Ablation-Inductively Coupled Plasma Mass Spectrometry, Anal. Chem. 79 (2007) 6874-6880]. Such improvement couldn't be explained solely by the difference of amount of material ablated, and then, was attributed to the aerosol properties. In order to validate this hypothesis, the characterization of the aerosol produced by nanosecond and high repetition rate femtosecond laser ablation of polyacrylamide gels was investigated. Our 2D high repetition rate femtosecond laser ablation strategy of 2-mm wide lane was found to produce aerosols of similar particle size distribution compared to nanosecond laser ablation of 0.12-mm wide lane, with 38% mass of particles < 1 µm. However, at high repetition rate, when the ablated surface was reduced, the particle size distribution was shifted toward thinner particle diameter (up to 77% for a 0.12-mm wide lane at 285 µm depth). Meanwhile, scanning electron microscopy was employed to visualize the morphology of the aerosol. In the case of larger ablation, the fine particles ejected from the sample were found to form agglomerates due to higher ablation rate and then higher collision probability. Additionally, investigations of the plasma temperature changes during the ablation demonstrated that the introduction of such amount of polyacrylamide gel particles had very limited impact on the ICP source (Δ T~ 25 ± 5 K). This suggests that the cohesion forces between the thin particles composing these large aggregates were weak enough to have negligible impact on the ICPMS detection.

  15. Concanavalin A-reactive protein of rabbit thymocyte plasma membranes: analysis by crossed immune electrophoresis and sodium dodecylsulfate/polyacrylamide gel electrophoresis.

    PubMed

    Schmidt-Ullrich, R; Wallach, D F; Hendricks, J

    1975-03-25

    1. Thymocyte plasma membrane extracts, prepared with the non-ionic detergent Triton X-100, show 10 major protein components upon sodium dodecysulfate/polyacrylamide gel electrophoresis and at least 11 immunologic components upon crossed immune electrophoresis. 2. Concanavalin A reactive membrane proteins have been identified using crossed immune electrophoresis with receptor-ligand interaction. 3. These proteins are absorbed from Triton X-100-solubilized membranes onto immobilized concanavalin A. They are eluted in stepwise fashion, using increasing concentrations of alpha-methyl-d-glucoside, between 0.0004 M and 0.1 M. The predominant proteins eluted in each step are components with high electrophoretic mobility in crossed immune electrophoresis and are identical with a glycosylated component in sodium dodecysulfate/polyacrylamide gel electrophoresis with molecular weight of 55 000. 4. This component forms multimers in the presence of Triton X-100 which are not totally dissociated in sodium dodecylsulfate. 5. Neuramidase treatment followed by crossed immune electrophoresis of total plasma membrane isolates, as well as the purified glycoprotein fraction, indicates that the concanavalin A-reactive proteins are sialoglycoproteins. 6. Sodium dodecylsulfate component 5.1 comprises at least two different populations of glycoproteins (6 and 9) in crossed immune electrophoresis, one of which exclusively exhibits heterogenous carbohydrate antigenic sites (component 9). 7. Present data, taken together with previously published experiments, indicate that concanavalin A binding to intact thymocytes induces an increased turnover and release of the receptor protein(s). PMID:1125237

  16. Filipin staining of lipoproteins in polyacrylamide gels: sensitivity and photobleaching of the fluorophore and its use in a double staining method.

    PubMed

    Smejkal, G B; Hoff, H F

    1994-07-01

    We have developed a double staining procedure in which polyacrylamide gels are first stained with filipin to identify lipoproteins, and then with Coomassie Brilliant Blue (CBB) to identify proteins. Filipin staining when performed at 37 degrees C is both more rapid and more sensitive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm3 of band volume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm3. A semilogarithmic relationship was found between the amount of LDL unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumination of these gels, such photobleaching actually resulted in maximizing of the signal:noise ratio, resulting in better definition of bands. Treatment of gels with filipin had no deleterious effects on the subsequent staining with CBB. This dual staining procedure should prove useful for studies in which both lipoproteins and proteins in plasma need to be documented in the same gel. PMID:7529172

  17. Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

    SciTech Connect

    Prem Das, O.; Henderson, E.J.

    1986-11-01

    Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum.

  18. On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin.

    PubMed

    Herr, Amy E; Throckmorton, Daniel J; Davenport, Andrew A; Singh, Anup K

    2005-01-15

    By integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device, we have developed a microanalytical platform for performing electrophoresis-based immunoassays. The microfluidic immunoassays are performed by gel electrophoretic separation and quantitation of bound and unbound antibody or antigen. To retain biological activity of proteins and maintain intact immune complexes, nondenaturing polyacrylamide gel electrophoresis conditions were investigated. Both direct (noncompetitive) and competitive immunoassay formats are demonstrated in microchips. A direct immunoassay was developed for detection of tetanus antibodies in buffer as well as diluted serum samples. After an off-chip incubation step, the immunoassay was completed in less than 3 min and the sigmoidal dose-response curve spanned an antibody concentration range from 0.17 to 260 nM. The minimum detectable antibody concentration was 0.68 nM. A competitive immunoassay was also developed for tetanus toxin C-fragment by allowing unlabeled and fluorescently labeled tetanus toxin C-fragment compete to bind to a limited fixed concentration of tetanus antibody. The immunoassay technique described in this work shows promise as a component of an integrated microfluidic device amenable to automation and relevant to development of clinical diagnostic devices. PMID:15649057

  19. Data on final calcium concentration in native gel reagents determined accurately through inductively coupled plasma measurements.

    PubMed

    Viviano, Jeffrey; Wu, Hao; Krishnan, Anuradha; Ramanujachary, Kandalam; Venkataraman, Venkat

    2016-03-01

    In this article we present data on the concentration of calcium as determined by Inductively Coupled Plasma (ICP) measurements. Calcium was estimated in the reagents used for native gel electrophoresis of Neuronal Calcium Sensor (NCS) proteins. NCS proteins exhibit calcium-dependent mobility shift in native gels. The sensitivity of this shift to calcium necessitated a precise determination of calcium concentrations in all reagents used. We determined the calcium concentrations in different components used along with the samples in the native gel experiments. These were: 20 mM Tris pH 7.5, loading dye and running buffer, with distilled water as reference. Calcium determinations were through ICP measurements. It was found that the running buffer contained calcium (244 nM) over the blank. PMID:26937454

  20. Data on final calcium concentration in native gel reagents determined accurately through inductively coupled plasma measurements

    PubMed Central

    Viviano, Jeffrey; Wu, Hao; Krishnan, Anuradha; Ramanujachary, Kandalam; Venkataraman, Venkat

    2016-01-01

    In this article we present data on the concentration of calcium as determined by Inductively Coupled Plasma (ICP) measurements. Calcium was estimated in the reagents used for native gel electrophoresis of Neuronal Calcium Sensor (NCS) proteins. NCS proteins exhibit calcium-dependent mobility shift in native gels. The sensitivity of this shift to calcium necessitated a precise determination of calcium concentrations in all reagents used. We determined the calcium concentrations in different components used along with the samples in the native gel experiments. These were: 20 mM Tris pH 7.5, loading dye and running buffer, with distilled water as reference. Calcium determinations were through ICP measurements. It was found that the running buffer contained calcium (244 nM) over the blank. PMID:26937454

  1. Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence.

    PubMed

    Kang, Chulhun; Kim, Hyun Jung; Kang, Donghoon; Jung, Duk Young; Suh, Myungkoo

    2003-10-01

    Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. PMID:14595675

  2. Polyacrylamide gel substrates that simulate the mechanical stiffness of normal and malignant neuronal tissues increase protoporphyin IX synthesis in glioma cells

    NASA Astrophysics Data System (ADS)

    Niu, Carolyn J.; Fisher, Carl; Scheffler, Kira; Wan, Rachel; Maleki, Hoda; Liu, Haijiao; Sun, Yu; Simmons, Craig A.; Birngruber, Reginald; Lilge, Lothar

    2015-09-01

    Protoporphyrin IX (PPIX) produced following the administration of exogenous 5d-aminolevulinic acid is clinically approved for photodynamic therapy and fluorescence-guided resection in various jurisdictions around the world. For both applications, quantification of PPIX forms the basis for accurate therapeutic dose calculation and identification of malignant tissues for resection. While it is well established that the PPIX synthesis and accumulation rates are subject to the cell's biochemical microenvironment, the effect of the physical microenvironment, such as matrix stiffness, has received little attention to date. Here we studied the proliferation rate and PPIX synthesis and accumulation in two glioma cell lines U373 and U118 cultured under five different substrate conditions, including the conventional tissue culture plastic and polyacrylamide gels that simulated tissue stiffness of normal brain (1 kPa) and glioblastoma tumors (12 kPa). We found that the proliferation rate increased with substrate stiffness for both cell lines, but not in a linear fashion. PPIX concentration was significantly higher in cells cultured on tissue-simulating gels than on the much stiffer tissue culture plastic for both cell lines. These findings, albeit preliminary, suggest that the physical microenvironment might be an important determinant of tumor aggressiveness and PPIX synthesis in glioma cells.

  3. System and method of infrared matrix-assisted laser desorption/ionization mass spectrometry in polyacrylamide gels

    DOEpatents

    Haglund, Jr., Richard F.; Ermer, David R.; Baltz-Knorr, Michelle Lee

    2004-11-30

    A system and method for desorption and ionization of analytes in an ablation medium. In one embodiment, the method includes the steps of preparing a sample having analytes in a medium including at least one component, freezing the sample at a sufficiently low temperature so that at least part of the sample has a phase transition, and irradiating the frozen sample with short-pulse radiation to cause medium ablation and desorption and ionization of the analytes. The method further includes the steps of selecting a resonant vibrational mode of at least one component of the medium and selecting an energy source tuned to emit radiation substantially at the wavelength of the selected resonant vibrational mode. The medium is an electrophoresis medium having polyacrylamide. In one embodiment, the energy source is a laser, where the laser can be a free electron laser tunable to generate short-pulse radiation. Alternatively, the laser can be a solid state laser tunable to generate short-pulse radiation. The laser can emit light at various ranges of wavelength.

  4. Light Scattering Induced Giant Red-Shift in Photoluminescence from CdTe Quantum Dots Encapsulated in Polyacrylamide Gel Nanospheres

    NASA Astrophysics Data System (ADS)

    Garner, Brett W.; Cai, Tong; Hu, Zhibing; Kim, Moon; Neogi, Arup

    2009-07-01

    The photoluminescence emission from CdTe quantum dots embedded in hydrogel nanospheres based on poly(N-isopropylacrylamide) (PNIPAM) polymer is observed to be modified by the random light scattering within the colloidal medium. Photoluminescence emission from CdTe quantum dots of various size has been observed making the gel fluorescent. The optical properties of the quantum dots entrapped within the gel microspheres can be modified due to change in refractive index, volume density of the surrounding hydrogel medium. A red-shift of ˜100 nm has been observed from quantum dots emitting in the green wavelength region as the cell length is increased. This shift is due to secondary scattering and energy transfer induced by the larger scattering cross-section within the medium which results in a re-excitation of larger sized quantum dots.

  5. Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels.

    PubMed

    Zhu, Wenhong; Venable, John; Giometti, Carol S; Khare, Tripti; Tollaksen, Sandra; Ahrendt, Angela J; Yates, John R

    2005-12-01

    2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation. PMID:16315175

  6. Detection of glycoproteins in polyacrylamide gels using Pro-Q Emerald 300 dye, a fluorescent periodate Schiff-base stain.

    PubMed

    Mehta-D'souza, Padmaja

    2012-01-01

    Pro-Q Emerald 300 glycoprotein stain generates a bright-green fluorescent signal upon reacting with periodic acid-oxidized carbohydrate groups on proteins. With this dye, it is possible to detect proteins directly in the gel without the need to transfer them to a membrane. This dye is more sensitive than the standard periodic acid Schiff's base which uses acidic fuchsin dye. PMID:22585521

  7. Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution.

    PubMed

    Steinberg, T H; Lauber, W M; Berggren, K; Kemper, C; Yue, S; Patton, W F

    2000-02-01

    SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry. PMID:10726749

  8. A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

    PubMed

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

    2015-10-01

    In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and β-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research. PMID:26177935

  9. Ion-enhanced fluorescence staining of sodium dodecyl sulfate-polyacrylamide gels using bis(8-p-toluidino-1-naphthalenesulfonate).

    PubMed

    Horowitz, P M; Bowman, S

    1987-09-01

    A method for the sensitive fluorescent staining of sodium dodecyl sulfate (SDS) gels that extends the applicability and sensitivity of existing procedures has been developed. SDS-protein complexes are able to bind the noncovalent hydrophobic probe, bis(8-p-toluidino-1-naphthalenesulfonate) (bisANS) with an increase in quantum yield that is considerably larger than that observed with the commonly used monomeric form, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The quantum yield of bisANS bound to the SDS-protein complex is greatly enhanced by incubation with one of a number of cations including potassium and barium. The use of bisANS with metal ion enhancements provides a method for staining SDS gels that can be more sensitive than commonly used methods based on the binding of Coomassie blue, and provides a simple and rapid method for the detection and quantitation of proteins. The use of metal ion enhancements also greatly increases the sensitivity of staining methods based on the use of 1,8-ANS. The present method is much more sensitive than previous noncovalent, flourescent, postelectrophoresis stains, but it retains their considerable advantages of speed, simplicity, and the ability to perform secondary procedures on the separated materials. PMID:3425913

  10. Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software.

    PubMed

    McEvoy, C R; Seshadri, R; Firgaira, F A

    1998-09-01

    We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed. PMID:9762444

  11. Temporal study of staphylococcal species on the skin of human subjects in isolation and clonal analysis of Staphylococcus capitis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    PubMed Central

    Maggs, A F; Pennington, T H

    1989-01-01

    The staphylococcal skin floras of an isolated group of subjects were studied for 1 year. A wide variation in isolation patterns was found for different species. Staphylococcus intermedius, previously thought to be of veterinary origin, was found to be part of the resident flora of some subjects, and this may indicate a wider role for it in clinical infection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S. capitis isolates indicated persistent skin colonization at some sites; each region was colonized by only one clone of that species, although an adjacent area could be inhabited by a separate clone. Nine clonal groups were identified by SDS-PAGE; there was a degree of specialization between the groups with regard to the sites which they colonized. The interaction between species at a single site was less well defined. Noncolonizing isolates often exhibited phenotypic similarities that were lower than expected when compared with their presumed source. The results of SDS-PAGE analysis were compared with immunoblotting, antibiograms, and biotyping, and SDS-PAGE analysis was found to be a useful and practical tool for epidemiological work. Images PMID:2687313

  12. Structural Studies of Avian Myeloblastosis Virus: Comparison of Polypeptides in Virion and Core Component by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    PubMed Central

    Stromberg, Kurt; Hurley, Nancy E.; Davis, Nancy L.; Rueckert, Roland R.; Fleissner, Erwin

    1974-01-01

    Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope. Images PMID:4129794

  13. Variation and Genomic Localization of Genes Encoding DROSOPHILA MELANOGASTER Male Accessory Gland Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    PubMed Central

    Whalen, Michael; Wilson, Thomas G.

    1986-01-01

    Accessory gland proteins from Drosophila melanogaster males have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into nine major bands. When individual males from 175 strains were examined, considerable polymorphism for nearly one-half of the major protein bands was seen, including null alleles for three bands. Variation was observed not only among long-established laboratory strains but also among stocks recently derived from natural populations. There was little difference in the amount of variation between P and M strains, indicating that P element mutagenesis is not a factor producing the variation. Codominant expression of variants for each of five bands was found in heterozygotes, suggesting structural gene variation and not posttranslational modification variation. Stocks carrying electrophoretic variants of four of the major proteins were used to map the presumed structural genes for these proteins; the loci were found to be dispersed on the second chromosome. Since males homozygous for variant proteins were fertile, the polymorphism seems to have little immediate effect on successful sperm transfer. We propose that a high degree of polymorphism can be tolerated because these proteins play a nutritive rather than enzymatic role in Drosophila reproduction. PMID:3095182

  14. Automated Genotyping of a Highly Informative Panel of 40 Short Insertion-Deletion Polymorphisms Resolved in Polyacrylamide Gels for Forensic Identification and Kinship Analysis

    PubMed Central

    Pena, Heloisa B.; Pena, Sérgio D. J.

    2012-01-01

    Objective Short insertion-deletion polymorphisms (indels) are the second most abundant form of genetic variations in humans after SNPs. Since indel alleles differ in size, they can be typed using the same methodological approaches and equipment currently utilized for microsatellite genotyping, which is already operational in forensic laboratories. We have previously shown that a panel of 40 carefully chosen indels has excellent potential for forensic identification, with combined probability of identity (match probability) of 7.09 × 10–17 for Europeans. Methods We describe the successful development of a multiplex system for genotyping the 40-indel panel in long thin denaturing polyacrylamide gels with silver staining. We also demonstrate that the system can be easily fully automated with a simple large scanner and commercial software. Results and Conclusion The great advantage of the new system of typing is its very low cost. The total price for laboratory equipment is less than EUR 10,000.-, and genotyping of an individual patient will cost less than EUR 10.- in supplies. Thus, the 40-indel panel described here and the newly developed ‘low-tech’ analysis platform represent useful new tools for forensic identification and kinship analysis in laboratories with limited budgets, especially in developing countries. PMID:22851937

  15. A rapid method of species identification of wild chironomids (Diptera: Chironomidae) via electrophoresis of hemoglobin proteins in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).

    PubMed

    Oh, J T; Epler, J H; Bentivegna, C S

    2014-10-01

    Studying aquatic benthic macroinvertebrates (BMIs) in the field requires accurate taxonomic identification, which can be difficult and time consuming. Conventionally, head capsule morphology has been used to identify wild larvae of Chironomidae. However, due to the number of species and possible damage and/or deformity of their head capsules, another supporting approach for identification is needed. Here, we provide hemoglobin (Hb) protein in hemolymph of chironomids as a new biomarker that may help resolve some of the ambiguities and difficulties encountered during taxonomic identification. Chironomids collected from two locations in Maine and New Jersey, USA were identified to the genus level and in some cases to the species-level using head capsule and body morphologies. The head capsule for a particular individual was then associated with a corresponding Hb protein profile generated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Distinct Hb profiles were observed from one group (Thienemannimyia) and four genera (Chironomus, Cricotopus, Dicrotendipes, and Glyptotendipes) of chironomids. Several species were polymorphic, having more than one Hb profile and/or having bands of the same size as those of other species. However, major bands and the combination of bands could distinguish individuals at the genus and sometimes species-level. Overall, this study showed that Hb profiles can be used in combination with head capsule morphology to identify wild chironomids. PMID:24923437

  16. Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    PubMed Central

    Schindler, J; Hynes, R; Hopkins, N

    1977-01-01

    We have sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the virion proteins of an N- and a B-tropic C-type virus derived from the BALB/c mouse and 21 putative recombinants, designated XLP-N viruses, obtained from seven crosses between these N- and B-tropic viruses. All the XLP-N viruses are N-tropic but posses the XC plaque morphology of their B-tropic virus parent. Three virion proteins, p15, p30, and gp70, of the parental viruses each differ in electrophoretic mobility. Two recombinants were found that possess a p15 that comigrates with p15 of the B virus; 19 possess a p15 that comigrates with N virus p15. Sixteen recombinants possess a gp70 that migrates like the gp70 of the B virus: four have gp70 with an electrophoretic mobility like that of the N virus gp70. All 21 recombinants possess a p30 that comigrates with p30 of their N virus parent. Given the origin and phenotype of XLP-N viruses, these results would seem to provide good evidence that these viruses are recombinants. Images PMID:197267

  17. 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. PMID:25957128

  18. Coordination mechanism, characterization, and photoluminescence properties of spinel ZnAl2O4 nanoparticles prepared by a modified polyacrylamide gel route

    NASA Astrophysics Data System (ADS)

    Sun, Guangzhuang; Sun, Guangai; Zhong, Mian; Wang, Shifa; Zu, Xiaotao; Xiang, Xia

    2016-03-01

    Single-phase ZnAl2O4 nanoparticles with the spinel structure were successfully synthesized using a modified polyacrylamide gel method according to the atomic ratio of Zn to Al = 1: 1.8. The as-prepared samples were characterized by means of X-ray powder diffraction (XRD), thermogravimetric analysis (TG), differential scanning calorimetry analysis (DSC), field-emission scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and photoluminescence (PL) spectra. XRD patterns show that the pure phase of ZnAl2O4 is obtained after heating the xerogel at 900°C for 5 h in air. The SEM images reveal that the ZnAl2O4 nanoparticles have a narrow particle size distribution and the average particle size is around 45 nm. Photoluminescence (PL) spectra demonstrate the single phase ZnAl2O4 nanoparticles have an emission peak located at 469 nm when excited by 350 nm light. The phase structure, coordination mechanism, and luminescence properties have been discussed on the basis of the experimental results.

  19. Development of methods for the charge-derivatization of peptides in polyacrylamide gels and membranes for their direct analysis using matrix-assisted laser desorption-ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Strahler, John R.; Smelyanskiy, Yanina; Lavine, Gary; Allison, John

    1997-12-01

    Approaches are being developed for the interfacing of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry with polyacrylamide gel electrophoresis, in which laser-irradiated samples desorb directly from a gel, or from a membrane on which gel-separated polypeptides have been transferred. Whether one- or two-dimensional electrophoretic separations have been performed, preparations for the MALDI experiment which follow must optimize detection of the analytes present and the generation of structural information. Procedures have been developed for forming charged derivatives of peptides in solution. When subjected to MALDI analysis, these charged derivatives produce ions in some cases where the underivatized peptide would not yield a response. The ions fragment following acceleration and yield informative and simple post-source decay (PSD) spectra. The development of approaches to interfacing this chemistry with MALDI directly from gels and membranes is presented here.

  20. Detection of human rotavirus by using an alkaline phosphatase-conjugated synthetic DNA probe in comparison with enzyme-linked immunoassay and polyacrylamide gel analysis.

    PubMed Central

    Olive, D M; Sethi, S K

    1989-01-01

    An alkaline phosphatase-conjugated synthetic oligodeoxyribonucleotide probe was compared with polyacrylamide gel electrophoresis (PAGE) detection of rotavirus RNA as well as an enzyme-linked immunosorbent assay (ELISA) for the detection of rotavirus in stools from young children with gastroenteritis. The synthetic probe did not cross-react with bacterial causative agents of diarrheal disease. Extraction of viral RNA from stool samples with a phenol-chloroform mixture was suitable for most samples. In some cases fecal pigments interfered with the reaction of the probe with viral RNA. The use of ion-exchange chromatography to further purify viral RNA removed contaminating pigments and increased the sensitivity of the probe assay. Of 260 stool specimens, 77 (30%) were positive for rotavirus when tested by PAGE analysis of rotavirus RNA. The synthetic probe identified 71 rotavirus specimens when RNA obtained by phenol-chloroform extraction followed by chromatographic purification was used (sensitivity, 91.0%; specificity, 96.7%). The ELISA results also agreed well with the electrophoretic analysis (sensitivity, 98.7%; specificity 94%) and the probe assay (sensitivity, 90%; specificity, 100%). Discordant results between the ELISA and the probe assay were examined further by electron microscopy and PAGE analysis of viral RNA. The positive and negative predictive values of the probe assay in comparison with PAGE were 92.2 and 96.1%, respectively. Rotaviruses showing both long and short RNA electrophoretic patterns were detected by the probe. The probe assay coupled with chromatographic purification of rotavirus RNA is an effective method for detecting rotavirus and compares favorably with PAGE analysis and ELISA. Images PMID:2536392

  1. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.

    PubMed Central

    Sonnenberg, M G; Belisle, J T

    1997-01-01

    A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis KatG catalase/peroxidase. Thus, the detailed mapping of M. tuberculosis proteins, combined with state-of-the-art analytical techniques such as mass spectrometry, provides a basis for further analysis and rapid identification of biologically relevant molecules. PMID:9353028

  2. Time-resolved Raman and polyacrylamide gel electrophoresis observations of nucleotide incorporation and misincorporation in RNA within a bacterial RNA polymerase crystal.

    PubMed

    Antonopoulos, Ioanna H; Murayama, Yuko; Warner, Brittany A; Sekine, Shun-Ichi; Yokoyama, Shigeyuki; Carey, Paul R

    2015-01-27

    The bacterial RNA polymerase (RNAP) elongation complex (EC) is highly stable and is able to extend an RNA chain for thousands of nucleotides. Understanding the processive mechanism of nucleotide addition requires detailed structural and temporal data for the EC reaction. Here, a time-resolved Raman spectroscopic analysis is combined with polyacrylamide gel electrophoresis (PAGE) to monitor nucleotide addition in single crystals of the Thermus thermophilus EC (TthEC) RNAP. When the cognate base GTP, labeled with (13)C and (15)N (*GTP), is soaked into crystals of the TthEC, changes in the Raman spectra show evidence of nucleotide incorporation and product formation. The major change is the reduction of *GTP's triphosphate intensity. Nucleotide incorporation is confirmed by PAGE assays. Both Raman and PAGE methods have a time resolution of minutes. There is also Raman spectroscopic evidence of a second population of *GTP in the crystal that does not become covalently linked to the nascent RNA chain. When this population is removed by "soaking out" (placing the crystal in a solution that contains no NTP), there are no perturbations to the Raman difference spectra, indicating that conformational changes are not detected in the EC. In contrast, the misincorporation of the noncognate base, (13)C- and (15)N-labeled UTP (*UTP), gives rise to large spectroscopic changes. As in the GTP experiment, reduction of the triphosphate relative intensity in the Raman soak-in data shows that the incorporation reaction occurs during the first few minutes of our instrumental dead time. This is also confirmed by PAGE analysis. Whereas PAGE data show *GTP converts 100% of the nascent RNA 14mer to 15mer, the noncognate *UTP converts only ∼50%. During *UTP soak-in, there is a slow, reversible formation of an α-helical amide I band in the Raman difference spectra peaking at 40 min. Similar to *GTP soak-in, *UTP soak-in shows Raman spectoscopic evidence of a second noncovalently bound *UTP population in the crystal. Moreover, the second population has a marked effect on the complex's conformational states because removing it by "soaking-out" unreacted *UTP causes large changes in protein and nucleic acid Raman marker bands in the time range of 10-100 min. The conformational changes observed for noncognate *UTP may indicate that the enzyme is preparing for proofreading to excise the misincorporated base. This idea is supported by the PAGE results for *UTP soak-out that show endonuclease activity is occurring. PMID:25584498

  3. Identification of protein complexes of microsomes in rat adipocytes by native gel coupled with LC-ESI-QTOF.

    PubMed

    Ke, Ming; Zhang, Yongqian; Xiong, Yan; Saeed, Yasmeen; Deng, Yulin

    2016-04-22

    The study of the composition of microsome proteins/complexes/interactions in adipocytes provides useful information for researchers related to energy metabolism disorders. The native gel coupled with LC-ESI-QTOF approach was employed here for separating protein complexes. We found a series of proteins functionally clustered in biological processes of protein metabolism, cellular carbohydrate catabolism, response to stimulus and wounding, macromolecular complex subunit organization, positive regulation of molecular function, regulation of programmed cell death and biomolecule transport. According to clustering of proteins' electrophoresis profiles across native gel fractions and bioinformatics data retrieval, protein complexes/interactions involved in protein metabolism, cellular carbohydrate catabolism, macromolecular complex subunit organization and biomolecule transport were identified. Besides, the results also revealed some functional linkages, which may provide useful information for discovering previously unknown interactions. The interaction between SSAO and ALDH2 was verified by co-immunoprecipitation. The native gel combining mass spectrometry approach appeared to be a useful tool for investigating microsome proteins and complexes to complement the traditional electrophoresis approaches. The native gel strategy together with our findings should facilitate future studies of the composition of rat adipocyte microsome protein complexes under different conditions. PMID:26886786

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

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel. PMID:23913286

  5. Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes, and human serum.

    PubMed

    Fiszer-Szafarz, B

    1984-11-15

    A gel electrophoretic technique which allows detection of hyaluronidase activity in the gel has been devised. The principle is that the high-molecular-weight substrate, hyaluronic acid, is included in the gel, where it cannot move in the electrical field. After the run, the gel is incubated under conditions allowing the enzyme to degrade the substrate. Upon staining with "Stains-all" dye (Eastman Kodak Co., 2718), zones of hyaluronidase activity appear as pink bands in a blue background. The sensitivity limit is less than 3 fkat equivalent to 2.2 NF mU. The method is applicable to all types of hyaluronidases and chondroitinase ABC. It enabled to be shown that some hyaluronidases are polymorphic. This technique also made it possible to detect easily hyaluronidase activity in normal human serum. This analytical method represents a convenient step in the purification of hyaluronidase. PMID:6084966

  6. Double staining of coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl sulfate-zinc reverse staining: sensitive detection of coomassie blue-undetected proteins.

    PubMed

    Fernandez-Patron, C; Hardy, E; Sosa, A; Seoane, J; Castellanos, L

    1995-01-01

    The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water (15 min, two times) and then subjected to imidazole-zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-staining gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinase, 47 kDa) over a wide range of protein molecular weights (10 to 100 kDa) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed. PMID:7535985

  7. A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

    PubMed

    Wiltfang, J; Arold, N; Neuhoff, V

    1991-05-01

    A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively. PMID:1718736

  8. Blue-native PAGE in plants: a tool in analysis of protein-protein interactions

    PubMed Central

    Eubel, Holger; Braun, Hans-Peter; Millar, A Harvey

    2005-01-01

    Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel-Electrophoresis (BN-PAGE). BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms, including bacteria, yeasts, animals and plants. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. PMID:16287510

  9. Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.

    PubMed

    Ladner, Carol L; Wishart, David S

    2012-07-01

    The formation of β-sheet-rich prion protein (PrP(β)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(β) complexes. PrP(β) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit. Calibration of the size of the PrP(β) oligomer bands was made possible with a cross-linked mouse PrP(90-232) ladder (1- to 11-mer) generated using ruthenium bipyridyl-based photoinduced cross-linking of unmodified proteins (PICUP). This PrP PICUP ladder allowed the size and abundance of PrP(β) oligomers formed from urea/salt and acid conversion to be determined. This distribution consists of 7-, 8-, 9-, 10-, and 11-mers, with the most abundant species being the 8-mer. The high-resolution separation afforded by RENAGE has allowed us to investigate distinctive size and population changes in PrP(β) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds. PMID:22490465

  10. Speciation analysis of inorganic arsenic in river water by Amberlite IRA 910 resin immobilized in a polyacrylamide gel as a selective binding agent for As(V) in diffusive gradient thin film technique.

    PubMed

    Rolisola, Ana M C M; Suárez, Carlos A; Menegário, Amauri A; Gastmans, Didier; Kiang, Chang H; Colaço, Camila D; Garcez, Daniel L; Santelli, Ricardo E

    2014-09-01

    In this study, a method is proposed for the selective retention of As(V) using diffusive gradient in thin film (DGT) samplers containing a strongly basic anion exchange resin (Amberlite IRA 910) supported on a polyacrylamide gel. In addition, the total arsenic content is determined by ferrihydrite gel discs. Subsequently, the concentration of As(III) was obtained by determining the difference between the total As and As(V). DGT experiments showed linear accumulation of As(V) (up to 280 ng) until a deployment time of 8 h deployment (R(2) > 0.99). The retention of As(V) was appropriate (97.9-112.3%) between pH 5 and 9. For a solution with an ionic strength ranging from 0.001 to 0.05 mol L(-1), the As(V) uptake ranged from 90-120%. The proposed method was applied for the speciation of arsenic in river water. For the analysis of spiked samples collected at the Furnas stream, the recoveries of total arsenic content ranged between 103.9% and 118.8%. However, the recoveries of As(III) and As(V) were 43.3-75.2% and 147.3-153.4%, respectively. These differences were probably because of the oxidation of As(III) to As(V) during deployments. For spiked samples collected at the Ribeirão Claro, the recoveries of dissolved As(III), As(V) and As(T) were 103.1%, 108.0% and 106.3%, respectively. Thus, the DGT technique with Amberlite IRA 910 resin as the binding phase can be employed for the in situ redox speciation of inorganic arsenic. PMID:25010156

  11. Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a (3H)estradiol binding assay based on isoelectric focusing in polyacrylamide gel

    SciTech Connect

    Pousette, A.; Gustafsson, S.A.; Thoernblad, A.M.N.; Nordgren, A.; Saellstroem, J.Li.; Lindgren, A.; Sundelin, P.; Gustafsson, J.A.

    1986-08-01

    Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of (/sup 3/H)estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.

  12. Gene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs.

    PubMed

    Medina-Suárez, R; Manning, K; Fletcher, J; Aked, J; Bird, C R; Seymour, G B

    1997-10-01

    mRNA was extracted from the pulp and peel of preclimacteric (d 0) bananas (Musa AAA group, cv Grand Nain) and those exposed to ethylene gas for 24 h and stored in air alone for a further 1 (d 2) and 4 d (d 5). Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products from the pulp and peel of these fruits revealed significant up-regulation of numerous transcripts during ripening. The majority of the changes were initiated by d 2, with the level of these messages increasing during the remainder of the ripening period. Pulp tissue from d 2 was used for the construction of a cDNA library. This library was differentially screened for ripening-related clones using cDNA from d-0 and d-2 pulp by a novel microtiter plate method. In the primary screen 250 up- and down-regulated clones were isolated. Of these, 59 differentially expressed clones were obtained from the secondary screen. All of these cDNAs were partially sequenced and grouped into families after database searches. Twenty-five nonredundant groups of pulp clones were identified. These encoded enzymes were involved in ethylene biosynthesis, respiration, starch metabolism, cell wall degradation, and several other key metabolic events. We describe the analysis of these clones and their possible involvement in ripening. PMID:9342865

  13. Measurement of tyrosine kinase activity in non-denaturing polyacrylamide gels and its induction during differentiation of human leukemia HL-60 cells

    SciTech Connect

    Glazer, R.I.; Chapekar, M.S.; Knode, M.C.

    1986-05-01

    The authors have developed a PAGE assay to measure tyrosine kinase (PK-T) activity in cell extracts. HL-60 cells were extracted sequentially with 0.1% and 1.0% Triton X-100 buffer to obtain soluble and membrane-bound proteins, respectively. Extracts were separated by PAGE at 4/sup 0/ and gels were incubated in the presence of the substrate Glu:Tyr (4:1) and an assay mixture containing Mn/sup + +/, Mg/sup + +/ and (..gamma../sup 32/P)ATP. Gels were washed in TCA:PPi, dried and autoradiographed. 0.1% Triton extracts of untreated cells in log phase displayed several PK-T activities of which the major species was approx.75 kD; 1% Triton extracts exhibited only residual 75 kD PK-T. In contrast, induction of differentiation in HL-60 cells with optimal concentrations of DMSO, retinoic acid, 1.25(OH)/sub 2/ vitamin D/sub 3/, phorbol ester, immune interferon (IFN-..gamma..) or tumor necrosis factor (TNF) induced the appearance of a approx.170 kD PK-T in the 1% Triton extracts and a reduction of the 75 kD PK-T in the 0.1% Triton extracts. The 170 kD PK-T could also utilize Glu:Tyr (6:3:1), histone H/sub 1/, pp60/sup src/ substrate Lys-14-Gly and vinculin as substrates but not Glu:Tyr (1:1). The 170 kD PK-T appeared within 2 days after treatment with 2500 ..mu../ml of IFN-..gamma.. or 100 ..mu../ml of TNF and its appearance coincided with the induction of the monocyte phenotype. These results suggest that the appearance of the 170 kD PK-T is related to the mature granulocyte or monocyte phenotype. Studies are in progress with radiolabeled IFN-..gamma.. to determine if the 170 kD PK-T is related to the IFN-..gamma.. receptor.

  14. EFFECTS OF NATIVE LIPID CONTENT ON THE GEL PROPERTIES AND SPHERULITE FORMATION OF JET COOKED CORNSTARCH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steam jet cooking is a process that has been used for many years to prepare aqueous starch dispersions for both food and industrial applications. In this process, gel properties of the final starch dispersion are dependent on several variables, including starch concentration, steam pressure, stirri...

  15. Preparation of DNA-crosslinked polyacrylamide hydrogels.

    PubMed

    Previtera, Michelle L; Langrana, Noshir A

    2014-01-01

    Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels. PMID:25226067

  16. Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies.

    PubMed

    Brown, Hilda H; Borchelt, David R

    2014-01-01

    Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1. PMID:25121776

  17. Analysis of Mutant SOD1 Electrophoretic Mobility by Blue Native Gel Electrophoresis; Evidence for Soluble Multimeric Assemblies

    PubMed Central

    Brown, Hilda H.; Borchelt, David R.

    2014-01-01

    Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1. PMID:25121776

  18. Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis.

    PubMed Central

    Hagler, J; Shuman, S

    1992-01-01

    We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway. Images PMID:1373199

  19. Predictive effect of XPA and XPD polymorphisms on survival of advanced NSCLC patients treated with platinum-based chemotherapy: a three-dimensional (3-D), polyacrylamide gel-based DNA microarray method.

    PubMed

    Cheng, Hongyan; Qin, Qin; Sun, Xinchen; Li, Fan; Sun, Ning; Cheng, Lu; Lu, Zuhong; Chen, Baoan

    2013-10-01

    Platinum-based chemotherapy is a primary treatment for patients with advanced non-small cell lung cancer (NSCLC). Considering individual differences, an effective and convenient method is urgently needed to identify the sensitivity of individual patient to platinum based regimen. Genetic variants in DNA repair genes are presumed to represent important determinants of drug efficacy. Our previous studies have demonstrated the involvement of xeroderma pigmentosum group A (XPA) codon23 and xeroderma pigmentosum group D (XPD) codon751 single-nucleotide polymorphisms (SNPs) in clinical response to platinum based chemotherapy in advanced NSCLC patients. Thus, a follow-up study was carried out to investigate the relevance of these genotypes and survival of the cohort (n = 115). The three-dimensional (3-D), polyacrylamide gel-based DNA microarray method was used to assess the genotypes of XPA and XPD in peripheral lymphocytes. Log-rank test revealed that the variant genotypes of XPA23 (A/G+G/G) were associated with significantly longer progression- free survival (PFS) (6.0 m vs. 10.6 m, log-rank P = 0.001) and overall survival (OS) (11.2 m vs. 20.8 m, log-rank P = 0.001). In Cox proportional hazards model, the hazard ratio (HR) for death in patients with G allele was 0.65 (P = 0.049). While no significant differences were observed in PFS or OS according to XPD Lys751Gln genotypes (log-rank P > 0.05). In combination with our previous short-term clinical results, this study further confirmed that by detecting the SNPs in blood cells, XPA A23G polymorphic variants might be a promising biomarker in predicting a favor prognosis of NSCLC patients and be helpful towards designing individualized treatments. PMID:23617284

  20. Two-dimensional blue native/SDS gel electrophoresis of multiprotein complexes from Helicobacter pylori.

    PubMed

    Pyndiah, Slovénie; Lasserre, Jean Paul; Ménard, Armelle; Claverol, Stéphane; Prouzet-Mauléon, Valérie; Mégraud, Francis; Zerbib, Frank; Bonneu, Marc

    2007-02-01

    The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins. PMID:17092930

  1. Microchannel gel electrophoretic separation systems and methods for preparing and using

    SciTech Connect

    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.

  2. Microchannel gel electrophoretic separation systems and methods for preparing and using

    SciTech Connect

    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.

  3. 2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: application to the analysis of IgG.

    PubMed

    Gonzalez, Leticia; Bustamante, Juan J; Barea-Rodriguez, Edwin J; Martinez, Andrew O; Haro, Luis S

    2006-05-01

    A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies. PMID:16703630

  4. Characterization of a gel-separated unknown glycoprotein by liquid chromatography/multistage tandem mass spectrometry: analysis of rat brain Thy-1 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    PubMed

    Itoh, Satsuki; Kawasaki, Nana; Harazono, Akira; Hashii, Noritaka; Matsuishi, Yukari; Kawanishi, Toru; Hayakawa, Takao

    2005-11-11

    We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins. PMID:16257296

  5. Analytical QbD: development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates.

    PubMed

    Pathak, Mili; Dutta, Debayon; Rathore, Anurag

    2014-08-01

    This paper presents a quality by design (QbD) based development of a novel native PAGE (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N-PAGE relevant to those academicians and other small companies involved in early-stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD-based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel. PMID:24643784

  6. Staining proteins in gels.

    PubMed

    Sasse, Joachim; Gallagher, Sean R

    2004-02-01

    This unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins. PMID:18432935

  7. White gels: an easy way to preserve methylene blue stained gels.

    PubMed

    Soto, Ana Maria; Draper, David

    2012-02-01

    Methylene blue can be used as a stain for visualizing nucleic acids in polyacrylamide gel electrophoresis. However, its relatively low sensitivity and reversible binding make it a temporary stain that diffuses from the gel relatively fast. Here we describe a very simple method for fixing methylene blue bands in nucleic acid polyacrylamide gels. The procedure makes the methylene blue stain permanent and increases the visibility of the bands, also contributing to increasing the sensitivity of methylene blue. PMID:22107889

  8. 21 CFR 172.255 - Polyacrylamide.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., Films and Related Substances § 172.255 Polyacrylamide. Polyacrylamide containing not more than 0.2 percent of acrylamide monomer may be safely used as a film former in the imprinting of soft-shell...

  9. Spring-loaded polymeric gel actuators

    DOEpatents

    Shahinpoor, Mohsen

    1995-01-01

    Spring-loaded electrically controllable polymeric gel actuators are disclosed. The polymeric gels can be polyvinyl alcohol, polyacrylic acid, or polyacrylamide, and are contained in an electrolytic solvent bath such as water plus acetone. The action of the gel is mechanically biased, allowing the expansive and contractile forces to be optimized for specific applications.

  10. Spring-loaded polymeric gel actuators

    DOEpatents

    Shahinpoor, M.

    1995-02-14

    Spring-loaded electrically controllable polymeric gel actuators are disclosed. The polymeric gels can be polyvinyl alcohol, polyacrylic acid, or polyacrylamide, and are contained in an electrolytic solvent bath such as water plus acetone. The action of the gel is mechanically biased, allowing the expansive and contractile forces to be optimized for specific applications. 5 figs.

  11. The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

    PubMed Central

    Cugno, Graziano; Parreira, José R.; Ferlizza, Enea; Hernández-Castellano, Lorenzo E.; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan

    2016-01-01

    Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss. PMID:27031334

  12. Investigations on preparation and properties of modified polyacrylamide hydrogels for application as wound dressing materials.

    PubMed

    Lukaszczyk, J

    1995-01-01

    Investigations on the influence of the polymerization conditions and feed ratio as well as various modification of the composite polyacrylamide-agar hydrogels on their properties has been presented. Results of the model studies have been utilized in the optimalization of the manufacturing parameters and procedures of the preparation of hydrogel dressing foils and granulated dressing gels. PMID:7479424

  13. Polyacrylamide phantom for self-actuating needle-tissue interaction studies.

    PubMed

    Datla, Naresh V; Konh, Bardia; Koo, Joe J Y; Choi, Daniel J W; Yu, Yan; Dicker, Adam P; Podder, Tarun K; Darvish, Kurosh; Hutapea, Parsaoran

    2014-01-01

    This study presents a polyacrylamide gel as a phantom material for needle insertion studies specifically developed for self-actuating needles to enhance the precise placement of needles in prostate. Bending of these self-actuating needles within tissue is achieved by Nitinol actuators attached to the needle body; however these actuators usually involve heating that can thermally damage the tissue surrounding the needles. Therefore, to develop and access feasibility of these needles, a polyacrylamide gel has been developed that mimics the thermal damage and mechanical properties of prostate tissue. Mechanical properties of the polyacrylamide gel was controlled by varying the concentrations of acrylamide monomer and N,N-methylene-bisacrylamide (BIS) cross-linker, and thermal sensitivity was achieved by adding bovine serum albumin (BSA) protein. Two polyacrylamide gels with different concentrations were developed to mimic the elastic modulus of the tissue. The two phantoms showed different rupture toughness and different deflection of bevel-tip needle. To study the thermal damage, a Nitinol wire was embedded in the phantom and resistively heated. The measured opaque zone (0.40mm) formed around the wire was close to the estimated damage zone (0.43mm) determined using the cumulative equivalent minutes at 43°C. PMID:23932314

  14. Templated Native Silk Smectic Gels

    NASA Technical Reports Server (NTRS)

    Jin, Hyoung-Joon (Inventor); Park, Jae-Hyung (Inventor); Valluzzi, Regina (Inventor)

    2013-01-01

    One aspect of the present invention relates to a method of preparing a fibrous protein smectic hydrogel by way of a solvent templating process, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; and collecting the resulting fibrous protein smectic hydrogel and allowing it to dry. Another aspect of the present invention relates to a method of obtaining predominantly one enantiomer from a racemic mixture, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; allowing the enantiomers of racemic mixture to diffuse selectively into the smectic hydrogel in solution; removing the smectic hydrogel from the solution; rinsing predominantly one enantiomer from the surface of the smectic hydrogel; and extracting predominantly one enantiomer from the interior of the smectic hydrogel. The present invention also relates to a smectic hydrogel prepared according to an aforementioned method.

  15. Templated native silk smectic gels

    NASA Technical Reports Server (NTRS)

    Jin, Hyoung-Joon (Inventor); Park, Jae-Hyung (Inventor); Valluzzi, Regina (Inventor)

    2009-01-01

    One aspect of the present invention relates to a method of preparing a fibrous protein smectic hydrogel by way of a solvent templating process, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; and collecting the resulting fibrous protein smectic hydrogel and allowing it to dry. Another aspect of the present invention relates to a method of obtaining predominantly one enantiomer from a racemic mixture, comprising the steps of pouring an aqueous fibrous protein solution into a container comprising a solvent that is not miscible with water; sealing the container and allowing it to age at about room temperature; allowing the enantiomers of racemic mixture to diffuse selectively into the smectic hydrogel in solution; removing the smectic hydrogel from the solution; rinsing predominantly one enantiomer from the surface of the smectic hydrogel; and extracting predominantly one enantiomer from the interior of the smectic hydrogel. The present invention also relates to a smectic hydrogel prepared according to an aforementioned method.

  16. Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.

    PubMed

    Mayer, E J; McKenna, E; Garsky, V M; Burke, C J; Mach, H; Middaugh, C R; Sardana, M; Smith, J S; Johnson, R G

    1996-01-19

    Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies. PMID:8576168

  17. Electrically controlled polymeric gel actuators

    DOEpatents

    Adolf, Douglas B.; Shahinpoor, Mohsen; Segalman, Daniel J.; Witkowski, Walter R.

    1993-01-01

    Electrically controlled polymeric gel actuators or synthetic muscles capable of undergoing substantial expansion and contraction when subjected to changing pH environments, temperature, or solvent. The actuators employ compliant containers for the gels and their solvents. The gels employed may be cylindrical electromechanical gel fibers such as polyacrylamide fibers or a mixture of poly vinyl alcohol-polyacrylic acid arranged in a parallel aggregate and contained in an electrolytic solvent bath such as salt water. The invention includes smart, electrically activated devices exploiting this phenomenon. These devices are capable of being manipulated via active computer control as large displacement actuators for use in adaptive structure such as robots.

  18. Electrically controlled polymeric gel actuators

    DOEpatents

    Adolf, D.B.; Shahinpoor, M.; Segalman, D.J.; Witkowski, W.R.

    1993-10-05

    Electrically controlled polymeric gel actuators or synthetic muscles are described capable of undergoing substantial expansion and contraction when subjected to changing pH environments, temperature, or solvent. The actuators employ compliant containers for the gels and their solvents. The gels employed may be cylindrical electromechanical gel fibers such as polyacrylamide fibers or a mixture of poly vinyl alcohol-polyacrylic acid arranged in a parallel aggregate and contained in an electrolytic solvent bath such as salt water. The invention includes smart, electrically activated devices exploiting this phenomenon. These devices are capable of being manipulated via active computer control as large displacement actuators for use in adaptive structure such as robots. 11 figures.

  19. A Simple Vertical Slab Gel Electrophoresis Apparatus.

    ERIC Educational Resources Information Center

    Carter, J. B.; And Others

    1983-01-01

    Describes an inexpensive, easily constructed, and safe vertical slab gel kit used routinely for sodium dodecyl sulphate-polyacrylamide gel electrophoresis research and student experiments. Five kits are run from a single transformer. Because toxic solutions are used, students are given plastic gloves and closely supervised during laboratory

  20. A Simple Vertical Slab Gel Electrophoresis Apparatus.

    ERIC Educational Resources Information Center

    Carter, J. B.; And Others

    1983-01-01

    Describes an inexpensive, easily constructed, and safe vertical slab gel kit used routinely for sodium dodecyl sulphate-polyacrylamide gel electrophoresis research and student experiments. Five kits are run from a single transformer. Because toxic solutions are used, students are given plastic gloves and closely supervised during laboratory…

  1. Staining proteins in gels.

    PubMed

    Sasse, Joachim; Gallagher, Sean R

    2003-08-01

    This unit describes protocols for detecting protein in a gel by either Coomassie blue staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins. PMID:18265316

  2. Evaluation du potentiel radiosensibilisateur ou radioprotecteur/antioxydant de quelques composes selectionnes par dosimetrie par gel de polyacrylamide et dosimetre de Fricke, et utilisation de la filamentation par impulsion laser infrarouge fenitoseconde comme un nouveau et puissant faisceau pour la radiotherapie du cancer

    NASA Astrophysics Data System (ADS)

    Meesat, Ridthee

    In radiation treatment, a sufficiently high radiation dose must be delivered to the tissue volumes containing the tumor cells while the lowest possible dose should be deposited in surrounding healthy tissue. We developed an original approach that is fast and easy to implement for the early assessment of the efficiency of radiation sensitizers and protectors. In addition, we characterized a new femtosecond laser pulse irradiation technique. We are able to deposit a considerable dose with a very high dose rate inside a well-controlled macroscopic volume without deposition of energy in front or behind the target volume. The radioprotective efficiency was measured by irradiation of the Fricke solution incorporating a compound under study and measuring the corresponding production of ferric ions G(Fe3+). The production of ferric ions is most sensitive to the radical species produced in the radiolysis of water. We studied experimentally and simulated with a full Monte-Carlo computer code the radiation-induced chemistry of Fricke/cystamine solutions. Results clearly indicate that the protective effect of cystamine originates from its radical-capturing ability, which allows this compound to compete with the ferrous ions for the various free radicals - especially ·OH radicals and H· atoms - formed during irradiation of the surrounding water. The sensitizing capacity of radiation sensitizers was measured by irradiation of a polyacrylamide gel (PAG) dosimeter incorporating a compound under study and measuring the corresponding increase in the gradient between spin-spin relaxation rate (R2) and absorbed dose. We measured an irradiation energy-dependent increase in R 2-dose sensitivity for halogenated compounds or a decrease for radioprotectors. Finally, we studied a novel laser irradiation method called "filamentation". We showed that this phenomenon results in an unprecedented deposition of energy and the dose rate thus achieved exceeds by orders of magnitude values previously reported for the most intense clinical radiotherapy systems. Moreover, the length of the dose-free entrance region was adjusted by selecting the duration of femtosecond laser pulses. In addition, we provided evidence that the biological damage caused by this irradiation was similar to other ionizing radiation sources. Keywords: Radiotherapy, radiosensitization, radioprotection, laser, filamentation, dosimetry.

  3. Crystallization of calcium phosphate in polyacrylamide hydrogels containing phosphate ions

    NASA Astrophysics Data System (ADS)

    Yokoi, Taishi; Kawashita, Masakazu; Kikuta, Koichi; Ohtsuki, Chikara

    2010-08-01

    Calcium phosphate crystals were formed in polyacrylamide (PAAm) hydrogels containing phosphate ions by diffusion of calcium ions from calcium nitrate (Ca(NO 3) 2) solutions covering the gels. Changes in crystalline phases and crystal morphology of calcium phosphate, and in ion concentrations of the Ca(NO 3) 2 solutions were investigated as a function of reaction time. Single or two coexisting crystalline phases of calcium phosphate, hydroxyapatite (HAp), HAp/dicalcium phosphate dihydrate (DCPD) or octacalcium phosphate (OCP)/DCPD were formed in the gels. HAp crystals are formed near the surface of the gels. The dense HAp layer and HAp/DCPD layer prevented diffusion of calcium ions from the Ca(NO 3) 2 solution, thus formation of calcium phosphate in the gel phase was inhibited. Formation of DCPD was observed to follow the formation of OCP or HAp. The size of the OCP crystals gradually increased with reaction time, while changes in size of HAp crystals were not observed. The reaction time required for DCPD formation depended on the degree of supersaturation with respect to DCPD in the systems. DCPD formed within 1 day under high supersaturation conditions, whereas it formed at 10 days in low supersaturation conditions.

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

    PubMed

    Krajewski, Wladyslaw A

    2016-03-15

    The solid-phase protein assays using blotting membranes as hard support do not allow achieving the low background and sensitivity of protein staining in clear gels. The membrane opacity complicates imaging of results on standard lab documentation systems. We describe a low-cost transparent matrix that can be used as an alternative to polymeric membranes for solid-phase assays. Protein samples are spotted onto a dry film of composite agarose-polyacrylamide matrix covering standard glass microscopic slides. After rehydration in protein-fixing solution, matrix with protein samples can be detached from glass support and stained as conventional protein polyacrylamide gels. PMID:26706799

  5. Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of Rhodococcus rhodochrous PA-34.

    PubMed

    Raj, J; Prasad, S; Sharma, N N; Bhalla, T C

    2010-09-01

    The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells. PMID:20941578

  6. Staining proteins in gels.

    PubMed

    Sasse, Joachim; Gallagher, Sean R

    2009-01-01

    This unit describes protocols for detecting protein in a gel by Coomassie blue, silver, or fluorescent staining. As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Alternate protocols describe rapid Coomassie and silver staining methods, as well as fluorescent stains that are specific for phosphoproteins and glycoproteins. Staining of proteins in SDS-polyacrylamide gels is described; variations for fluorescent staining of proteins in nondenaturing gels are also included. Support protocols describe photography of stained proteins. PMID:19170026

  7. Autofluorescence based visualization of proteins from unstained native-PAGE

    NASA Astrophysics Data System (ADS)

    Manjunath, S.; Rao, Bola Sadashiva S.; Satyamoorthy, Kapaettu; Mahato, Krishna Kishore

    2015-03-01

    Proteins are the most diverse and functionally active biomolecules in the living system. In order to understand their diversity and dynamic functionality, visualization in native form without altering structural and functional properties during the separation from the complex mixtures is very much essential. In the present study, a sensitive methodology for optimal visualization of unstained or untagged proteins in native poly-acrylamide gel electrophoresis (N-PAGE) has been developed where, concentration of the acrylamide and bis-acrylamide mixture, Percentage of the gel, fixing of the N-PAGE by methanol: acetic acid: water and washing of the gel in the mili-Q water has been optimized for highest sensitivity using laser induced autofluorescence. The outcome with bovine serum albumin (BSA) in PAGE was found to be highest at acrylamide and bis-acrylamide concentrations of 29.2 and 0.8 respectively in 12% N-PAGE. After the electrophoresis run, washing of the N-PAGE immediately with miliQ water for 12 times and eliminating the methanol: acetic acid: water, fixing of the N-PAGE yielded better sensitivity of visualization. Using the above methodology 25ng of BSA protein band in PAGE was clearly identified by the technique. The currently used staining techniques for the visualization of proteins are coomassie brilliant blue and silver staining, have the sensitivity of 100ng and 5ng respectively. The current methodology was found to be more sensitive as compared to coomassie staining and less sensitive compared to silver staining respectively. The added advantage of this methodology is the faster visualization of proteins without altering their structure and functional properties.

  8. 21 CFR 172.255 - Polyacrylamide.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Polyacrylamide. 172.255 Section 172.255 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION...

  9. Polyacrylamide Hydrogel Properties for Horticultural Applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyacrylamide (PAAm) hydrogels are commonly employed to ensure hydration of the growth media and minimize crop losses during the crop production and postproduction phases in horticulture. However, studies of the effect of these materials have shown that they have a minimal effect on crop life and q...

  10. 21 CFR 173.10 - Modified polyacrylamide resin.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... conditions: (a) The modified polyacrylamide resin is produced by the copolymerization of acrylamide with not... polyacrylamide resin contains not more than 0.05 percent residual acrylamide. (c) The modified...

  11. Conducting Polymer Electrodes for Gel Electrophoresis

    PubMed Central

    Bengtsson, Katarina; Nilsson, Sara; Robinson, Nathaniel D.

    2014-01-01

    In nearly all cases, electrophoresis in gels is driven via the electrolysis of water at the electrodes, where the process consumes water and produces electrochemical by-products. We have previously demonstrated that π-conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) can be placed between traditional metal electrodes and an electrolyte to mitigate electrolysis in liquid (capillary electroosmosis/electrophoresis) systems. In this report, we extend our previous result to gel electrophoresis, and show that electrodes containing PEDOT can be used with a commercial polyacrylamide gel electrophoresis system with minimal impact to the resulting gel image or the ionic transport measured during a separation. PMID:24586761

  12. Performance of 18 polymers in aluminum citrate colloidal dispersion gels

    SciTech Connect

    Smith, J.E.

    1995-11-01

    Colloidal dispersion gels are made up of low concentrations of polymer and aluminum citrate in water. These gels, which are mixed as a homogeneous solution at the surface, provide a valuable tool for in-depth blockage of high permeability regions of rock in heterogeneous reservoirs. Performance of colloidal dispersion gels depends strongly on the type and quality of polymer used. This paper provides an overview of the performance of 18 different polymers in colloidal dispersion gels. 14 of the polymers were partially hydrolyzed polyacrylamides or AMPS polymers in dry crystalline form with varying degrees of hydrolysis and molecular weight. The group also includes one cationic polyacrylamide, one carboxymethyl cellulose, one partially hydrolyzed polyacrylamide in emulsion form and one polysaccharide in dry form. Gels were mixed with the polymers at two polymer concentrations, three polymer:aluminum ratios and in different concentrations of potassium chloride. The gels were quantitatively tested at 1, 7, 14 and 28 days after crosslinking using the transition pressure test, which is a screen flow resistance test. Of the six polymer types tested, only the dry partially hydrolyzed polyacrylamides and AMPS polymers formed colloidal dispersion gels. Gel strength generally increased with increasing anionic charge and molecular weight; however, the manner in which the polymer is manufactured and the impurities present in the polymer also play roles which are more significant than originally expected.

  13. Acetyl CoA Carboxylase: Isolation and Characterization of Native Biotin Carboxyl Carrier Protein

    PubMed Central

    Fall, R. Ray; Nervi, A. M.; Alberts, Alfred W.; Vagelos, P. Roy

    1971-01-01

    A large form of biotin carboxyl carrier protein (BCCPL) has been isolated from extracts of Escherichia coli. It has a minimal molecular weight of 20,000, according to its behavior on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and contains approximately 1 mol of biotin per 22,000 g of protein. BCCPL exhibits Km values, in the biotin carboxylase and transcarboxylase half-reactions of acetyl CoA carboxylase, of 2 × 10-7 M and 4 × 10-7 M, respectively; these values are 50-100 times lower than those obtained with smaller forms of BCCP previously isolated. Electrophoresis of crude extracts of E. coli indicates that the major biotin-containing protein migrates at the same rate as BCCPL, which suggests that BCCPL is the native form of BCCP in E. coli. Images PMID:4934522

  14. 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. PMID:22585527

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

    PubMed

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

    2015-07-15

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis. PMID:25908558

  16. Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE)

    PubMed Central

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

    2015-01-01

    The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria. PMID:25859224

  17. EVALUATION OF NOVEL PRECAST SDS-PAGE GELS FOR SEPARATION OF SORGHUM PROTEINS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pre-cast polyacrylamide gels using novel buffer chemistry for enhanced resolution and shelf life stability were evaluated for separating sorghum proteins. Two gels with different acrylamide concentrations, 12 and 4-12%, were tested with two different buffer systems. Gels were evaluated for separat...

  18. Adsorption characteristics of polyacrylamide and sulfonate-containing polyacrylamide copolymers on sodium kaolinite. [Gamma radiation

    SciTech Connect

    Hollander, A.F.; Somasundaran, P.; Gryte, C.C.

    1981-07-01

    Polyacrylamide and its copolymer containing 6.8 mole % 2-acrylamido-2-methylpropane sulfonic acid were prepared by an irradiation-initiated precipitation polymerization technique. The polymer was characterized by intrinsic viscosity under conditions similar to those used during adsorption measurements. Hydrolytic degradation of the polyacrylamide was found to be negligible under conditions used. The adsorption substrate, sodium kaolinite, was prepared by extensive ion exchange treatment. Equilibrium adsorption of the polymers on the sodium kaolinite was made as a function of polymer concentration, solution pH, ionic strength, and temperature.

  19. A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates.

    PubMed

    Krajewski, Wladyslaw A

    2015-02-01

    A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional "on-filter" assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of "on-membrane" staining with the sensitivity and ease of documentation of "in-gel" staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required. PMID:25449300

  20. Radio-synthesized polyacrylamide hydrogels for proteins release

    NASA Astrophysics Data System (ADS)

    Ferraz, Caroline C.; Varca, Gustavo H. C.; Lopes, Patricia S.; Mathor, Monica B.; Lugão, Ademar B.

    2014-01-01

    The use of hydrogels for biomedical purposes has been extensively investigated. Pharmaceutical proteins correspond to highly active substances which may be applied for distinct purposes. This work concerns the development of radio-synthesized hydrogel for protein release, using papain and bovine serum albumin as model proteins. The polymer was solubilized (1% w/v) in water and lyophilized. The proteins were incorporated into the lyophilized polymer and the hydrogels were produced by simultaneous crosslinking and sterilization using γ-radiation under frozen conditions. The produced systems were characterized in terms of swelling degree, gel fraction, crosslinking density and evaluated according to protein release, bioactivity and cytotoxicity. The hydrogels developed presented different properties as a function of polymer concentration and the optimized results were found for the samples containing 4-5% (w/v) polyacrylamide. Protein release was controlled by the electrostatic affinity of acrylic moieties and proteins. This selection was based on the release of the proteins during the experiment period (up to 50 h), maintenance of enzyme activity and the nanostructure developed. The system was suitable for protein loading and release and according to the cytotoxic assay it was also adequate for biomedical purposes, however this method was not able to generate a matrix with controlled pore sizes.

  1. A model of ideal elastomeric gels for polyelectrolyte gels.

    PubMed

    Li, Jianyu; Suo, Zhigang; Vlassak, Joost J

    2014-04-21

    The concept of the ideal elastomeric gel is extended to polyelectrolyte gels and verified using a polyacrylamide-co-acrylic acid hydrogel as a model material system. A comparison between mixing and ion osmosis shows that the mixing osmosis is larger than the ion osmosis for small swelling ratios, while the ion osmosis dominates for large swelling ratios. We show further that the non-Gaussian chain effect becomes important in the elasticity of the polymer network at the very large swelling ratios that may occur under certain conditions of pH and salinity. We demonstrate that the Gent model captures the non-Gaussian chain effect well and that it provides a good description of the free energy associated with the stretching of the network. The model of ideal elastomeric gels fits the experimental data very well. PMID:24647731

  2. Breast cancer following polyacrylamide hydrogel injection for breast augmentation: A case report

    PubMed Central

    CHEN, GANG; WANG, YUJIA; HUANG, JIN-LONG

    2016-01-01

    Polyacrylamide hydrogel (PAAG) has been used for several years as an injectable implant for augmentation mammoplasty in China. Although patients who received PAAG injections experienced a number of complications, breast cancer following PAAG injection has been reported only in two cases. In this report, we present a case of breast cancer following PAAG injection for breast augmentation. Our study demonstrated that PAAG injection may increase the risk of breast cancer development. Early-stage breast cancer detection is difficult, since the breast is covered with the indurated injected gel. Thus, PAAG injection for augmentation mammoplasty may negatively affect breast cancer diagnosis and prognosis. PMID:26998299

  3. Data of microstructure and mechanical properties of carbon foams derived from sucrose/polyacrylamide hydrogel.

    PubMed

    Yao, Yao; Chen, Fei; Chen, Xi; Shen, Qiang; Zhang, Lianmeng

    2016-06-01

    An easy method that combined gel casting and physical foaming was used to fabricate modified carbon foams. The design of carbon foams from sucrose/polyacrylamide hydrogel is a new concept for controlling the microstructure and improving the compressive properties of carbon foams. This article provides the micrographs obtained from optical and scanning electron microscope for foaming solution and carbon foams. Weight loss data used to construct the thermo-gravimetric curves are included. Load-displacement data constructing the stress-strain curves and the derived compressive properties are also included. PMID:26933668

  4. Data of microstructure and mechanical properties of carbon foams derived from sucrose/polyacrylamide hydrogel

    PubMed Central

    Yao, Yao; Chen, Fei; Chen, Xi; Shen, Qiang; Zhang, Lianmeng

    2016-01-01

    An easy method that combined gel casting and physical foaming was used to fabricate modified carbon foams. The design of carbon foams from sucrose/polyacrylamide hydrogel is a new concept for controlling the microstructure and improving the compressive properties of carbon foams. This article provides the micrographs obtained from optical and scanning electron microscope for foaming solution and carbon foams. Weight loss data used to construct the thermo-gravimetric curves are included. Load–displacement data constructing the stress–strain curves and the derived compressive properties are also included. PMID:26933668

  5. 21 CFR 872.3420 - Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... polyacrylamide polymer denture adhesive. 872.3420 Section 872.3420 Food and Drugs FOOD AND DRUG ADMINISTRATION....3420 Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive. (a) Identification. A carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive is a...

  6. 21 CFR 872.3420 - Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... polyacrylamide polymer denture adhesive. 872.3420 Section 872.3420 Food and Drugs FOOD AND DRUG ADMINISTRATION....3420 Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive. (a) Identification. A carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive is a...

  7. 21 CFR 872.3420 - Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... polyacrylamide polymer denture adhesive. 872.3420 Section 872.3420 Food and Drugs FOOD AND DRUG ADMINISTRATION....3420 Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive. (a) Identification. A carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive is a...

  8. 21 CFR 872.3420 - Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... polyacrylamide polymer denture adhesive. 872.3420 Section 872.3420 Food and Drugs FOOD AND DRUG ADMINISTRATION....3420 Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive. (a) Identification. A carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive is a...

  9. 21 CFR 872.3420 - Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... polyacrylamide polymer denture adhesive. 872.3420 Section 872.3420 Food and Drugs FOOD AND DRUG ADMINISTRATION....3420 Carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive. (a) Identification. A carboxymethylcellulose sodium and cationic polyacrylamide polymer denture adhesive is a...

  10. Silver staining of 2D electrophoresis gels.

    PubMed

    Rabilloud, Thierry

    2012-01-01

    Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It -combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed. PMID:22665294

  11. Topological effects on viscoelasticity of polyacrylamide hydrogels

    NASA Astrophysics Data System (ADS)

    Kalfus, Jan; Lesser, Alan

    2011-03-01

    Viscoelastic behavior of long linear chains in a concentrated solution is governed by the topology of the molecules and interchain excluded volume interaction. As a consequence, chain diffusive motion is significantly retarded and such an assembly of chains exhibits highly pronounced entropy elastic behavior. In this contribution, two types of additional chain confinements imposed on a concentrated solution of linear polyacrylamide (L-PAA) will be discussed. The confinement was realized either by adding silica nano-filler into the concentrated solution of L-PAA or by cross-linking of acrylamide in the concentrated solution of L-PAA. While in the first case the trapped entanglement interaction is caused by interaction of chains with large nano-filler surface, in the second case the L-PAA chains are trapped among the cross-links of the PAA network. Viscoelastic response of both types of composite systems exhibited generic characteristics. In both cases, the trapped entanglement interaction significantly changed the relaxation spectrum of the matrix polymer solution and considerably enhanced the linear elastic modulus.

  12. Field demonstration of in situ grouting of radioactive solid waste burial trenches with polyacrylamide. [Polyacrylamide

    SciTech Connect

    Spalding, B.P.; Fontaine, T.A.

    1990-01-01

    Demonstrations of in situ grouting with polyacrylamide were carried out on two undisturbed burial trenches and one dynamically compacted burial trench in Solid Waste Storage Area (SWSA) 6 at Oak Ridge National Laboratory (ORNL). The injection of polyacrylamide was achieved quite facilely for the two undisturbed burial trenches which were filled with grout, at typical pumping rates of 95 L/min, in several batches injected over several days. The compacted burial trench, however, failed to accept grout at more than 1.9 L/min even when pressure was applied. Thus, it appears that burial trenches, stabilized by dynamic compaction, have a permeability too low to be considered groutable. The water table beneath the burial trenches did not respond to grout injections indicating a lack of hydrologic connection between fluid grout and the water table which would have been observed if the grout failed to set. Because grout set times were adjusted to less than 60 min, the lack of hydrologic connection was not surprising. Postgrouting penetration testing revealed that the stability of the burial trenches was increased from 26% to 79% that measured in the undisturbed soil surrounding the trenches. In situ permeation tests on the grouted trenches indicated a significant reduction in hydraulic conductivity of the trench contents from a mean of 2.1 {times} 10{sup {minus}3} to 1.85 {times} 10{sup {minus}5} cm/s. Preliminary observations indicated that grouting with polyacrylamide is an excellent method for both improved stability and hydrologic isolation of radioactive waste and its incidental hazardous constituents.

  13. Use of recombinant DNA derived human relaxin to probe the structure of the native protein

    SciTech Connect

    Canova-Davis, E.; Kessler, T.J.; Lee, P.J.; Fie, D.T.W.; Griffin, P.; Stults, J.T.; Rinderknecht, E. ); Wade, J.D. )

    1991-06-18

    This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was euqipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the completer amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (Cys{sup A10}-Cys{sup A15}, Cys{sup A11}-Cys{sup B11}, and Cys{sup A24}-Cys{sup B23}) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.

  14. In situ grouting of buried transuranic waste with polyacrylamide

    SciTech Connect

    Spalding, B.P.; Lee, S.Y.; Farmer, C.D.; Hyder, L.K.; Supaokit, P.

    1987-01-01

    This project is a demonstration and evaluation of the in situ hydrologic stabilization of buried transuranic waste at a humid site via grout injection. Two small trenches, containing buried transuranic waste, were filled with 34.000 L of polyacrylamide grout. Initial field results have indicated that voids within the trenches were totally filled by the grout and that the intratrench hydraulic conductivity was reduced to below field-measurable values. No evidence of grout constituents were observed in twelve perimeter groundwater monitoring wells indicating that grout was contained completely within the two trenches. Polyacrylamide grout was selected for field demonstration over the polyacrylate grout due to its superior performance in laboratory degradation studies. Also supporting the selection of polyacrylamide was the difficulty in controlling the set time of the acrylate polymerization. Based on preliminary degradation monitoring, the polyacrylamide was estimated to have a microbiological half-life of 362 years in the test soil. 15 refs., 9 figs., 12 tabs.

  15. Antioxidant effect of green tea on polymer gel dosimeter

    NASA Astrophysics Data System (ADS)

    Samuel, E. J. J.; Sathiyaraj, P.; Deena, T.; Kumar, D. S.

    2015-01-01

    Extract from Green Tea (GTE) acts as an antioxidant in acrylamide based polymer gel dosimeter. In this work, PAGAT gel was used for investigation of antioxidant effect of GTE.PAGAT was called PAGTEG (Polyacrylamide green tea extract gel dosimeter) after adding GTE. Free radicals in water cause pre polymerization of polymer gel before irradiation. Polyphenols from GTE are highly effective to absorb the free radicals in water. THPC is used as an antioxidant in polymer gel dosimeter but here we were replaced it by GTE and investigated its effect by spectrophotometer. GTE added PAGAT samples response was lower compared to THPC added sample. To increase the sensitivity of the PAGTEG, sugar was added. This study confirmed that THPC was a good antioxidant for polymer gel dosimeter. However, GTE also can be used as an antioxidant in polymer gel if use less quantity (GTE) and add sugar as sensitivity enhancer.

  16. Experimental investigation of the properties of polyelectrolyte gel

    NASA Astrophysics Data System (ADS)

    Pyschnyi, S.; Rydzewski, R.

    1991-03-01

    A polyelectrolyte gel, e.g. polyacrylamide gel has interesting thermo-mechanical properties. It swells in solution, increasing its volume by large factors and the swelling may be induced by a change of temperature, by the acetone content of the solution or by its acidity. The swelling can be reversed, i.e. the gel may be shrunk back to its original shape. The preparation of the gel is described here and quantitative details of the swelling or shrinking are reported. Particular emphasis is placed on the effect of a load on the properties of the gel. It is shown that a tensile load significantly affects the volume changes induced by changes of acidity of the solution. Some peculiar properties of the gel emerged during the investigation, such as anisotropy in thin gels and a decrease of the electrical resistance as an immediate consequence of both loading and unloading.

  17. Effect of cationic polyacrylamide on the processing and properties of nanocellulose films.

    PubMed

    Raj, Praveena; Varanasi, Swambabu; Batchelor, Warren; Garnier, Gil

    2015-06-01

    The use of high molecular weight cationic polyacrylamide (CPAM) was investigated to accelerate the drainage of nanocellulose (Microfibrillated Cellulose) suspensions into films. The mechanism was quantified and optimized by measuring the gel point, the lowest solids concentration at which a continuous network is formed. The flocculation of MFC was analysed as a function of the polyelectrolyte dosage, charge density and molecular weight as well as process parameters (drainage time) and material properties. The adsorption isotherms of CPAMs on nanocellulose and their zeta potential curves were also analysed as a function of CPAM charge and dosage. Measured CPAM adsorption capacities for the 50% and 10% charged 13MDa CPAM onto MFC were 5mg/g and 8mg/g, respectively, corresponding to adsorption coverage on cellulose of 0.14mg/m(2) and 0.22mg/m(2). The floc strength and drainability of MFC suspensions were quantified with the gel point as a function of CPAM properties. For all combinations of polyelectrolyte molecular weight and charge density, the gel point of a nanocellulose suspension goes through a minimum with increasing polymer dosage. The minimum gel point was independent of the polyelectrolyte charge density at constant molecular weight. However, it reduced with decreasing CPAM molecular weight, at a constant addition rate. The drainage time of a nanocellulose suspension into a film is reduced by 2/3 by halving the gel point from 0.2 to 0.1kg/m(3); this is due to the more flocculated suspension facilitating drainage between flocs. Nanocellulose films of increased porosity also result from reducing the gel point, signifying that the more open 3D structure of the flocculated cellulose suspension is retained upon drying the 2D film cellulose film structure. PMID:25702868

  18. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    PubMed

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins. PMID:25770625

  19. Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes.

    PubMed

    Kamemura, K; Kato, S

    1998-05-01

    A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction. This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe. All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis. This method was used to detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum. These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples. PMID:9570845

  20. Polyelectrolyte gels

    SciTech Connect

    Segalman, D.J.; Witkowski, W.R.

    1995-06-01

    Polyelectrolyte (PE) gels are swollen polymer/solvent networks that undergo a reversible volume collapse/expansion through various types of stimulation. Applications that could exploit this large deformation and solvent expulsion/absorption characteristics include robotic {open_quotes}fingers{close_quotes} and drug delivery systems. The goals of the research were to first explore the feasibility of using the PE gels as {open_quotes}smart materials{close_quotes} - materials whose response can be controlled by an external stimulus through a feedback mechanism. Then develop a predictive capability to simulate the dynamic behavior of these gels. This involved experimentally characterizing the response of well-characterized gels to an applied electric field and other stimuli to develop an understanding of the underlying mechanisms which cause the volume collapse. Lastly, the numerical analysis tool was used to simulate various potential engineering devices based on PE gels. This report discusses the pursuit of those goals through experimental and computational means.

  1. Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis.

    PubMed

    Zarei, Mohammad; Ahmadzadeh, Hossein; Goharshadi, Elaheh K; Farzaneh, Ali

    2015-08-01

    Here, we show, for the first time, the use of graphitic carbon nitride (g-C3N4) nanosheets to improve the resolution and efficiency of protein separation in gel electrophoresis. By loading 0.04% (m/v) g-C3N4 nanosheets into the polyacrylamide gel at 25 °C, the thermal conductivity increased approximately 80% which resulted in 20% reduction in Joule heating and overall increase of separation efficiency. Also, polymerization of acrylamide occurred in the absence of tetramethylethylenediamine (TEMED) when the polyacrylamide gel contained g-C3N4 nanosheets. Hence, the g-C3N4 act simultaneously as a polymerization catalyst as well as heat sinks to lower Joule heating effect on band broadening. PMID:26320809

  2. Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis.

    PubMed

    Guo, Peng; Yuan, Yasheng; Chi, Fanglu

    2014-09-01

    We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. PMID:25063162

  3. Site blocking effects on adsorbed polyacrylamide conformation

    NASA Astrophysics Data System (ADS)

    Brotherson, Brett A.

    The use of polymers as flocculating additives is a common practice in many manufacturing environments. However, exactly how these polymers interact with surfaces is relatively unknown. One specific topic which is thought to be very important to flocculation is an adsorbed polymer's conformation. Substantial amounts of previous work, mainly using simulations, have been performed to elucidate the theory surrounding adsorbed polymer conformations. Yet, there is little experimental work which directly verifies current theory. In order to optimize the use of polymer flocculants in industrial applications, a better understanding of an adsorbed polymer's conformation on a surface beyond theoretical simulations is necessary. This work looks specifically at site blocking, which has a broad impact on flocculation, adsorption, and surface modification, and investigated its effects on the resulting adsorbed polymer conformation. Experimental methods which would allow direct determination of adsorbed polymer conformational details and be comparable with previous experimental results were first determined or developed. Characterization of an adsorbed polymer's conformation was then evaluated using dynamic light scattering, a currently accepted experimental technique to examine this. This commonly used technique was performed to allow the comparison of this works results with past literature. Next, a new technique using atomic force microscopy was developed, building on previous experimental techniques, to allow the direct determination of an adsorbed polymer's loop lengths. This method also was able to quantify changes in the length of adsorbed polymer tails. Finally, mesoscopic simulation was attempted using dissipative particle dynamics. In order to determine more information about an adsorbed polymer's conformation, three different environmental factors were analyzed: an adsorbed polymer on a surface in water, an adsorbed polymer on a surface in aqueous solutions of varying ionic strength, and an adsorbed polymer on a surface functionalized with site blocking additives. This work investigated these scenarios using a low charge density high molecular weight cationic polyacrylamide. Three different substrates, for polymer adsorption were analyzed: mica, anionic latex, and glass. It was determined that, similar to previous studies, the adsorbed polymer layer thickness in water is relatively small even for high molecular weight polymers, on the order of tens of nanometers. The loop length distribution of a single polymer, experimentally verified for the first time, revealed a broad span of loop lengths as high as 1.5 microns. However, the bulk of the distribution was found between 40 and 260 nanometers. For the first time, previous theoretical predictions regarding the salt effect on adsorbed polymer conformation were confirmed experimentally. It was determined that the adsorbed polymer layer thickness expanded with increasing ionic strength of the solvent. Using atomic force microscopy, it was determined that the adsorbed polymer loop lengths and tail lengths increased with increasing ionic strength, supporting the results found using dynamic light scattering. The effect of the addition of site blocking additives on a single polymer's conformation was investigated for the first time. It was determined that the addition of site blocking additives caused strikingly similar results as the addition of salt to the medium. The changes in adsorbed polymer's loop lengths was found to be inconsistent and minimal. However, the changes in an adsorbed polymer's free tail length was found to increase with increasing site blocking additive levels. These results were obtained using either PDADMAC or cationic nanosilica as site blocking additives.

  4. Improving gel properties of hairtail surimi by electron irradiation

    NASA Astrophysics Data System (ADS)

    Lin, Xianping; Yang, Wenge; Xu, Dalun; Jie, Zhen; Liu, Wen

    2015-05-01

    Hairtail surimi was subjected to electron irradiation for doses up to 9 kGy. At 7 kGy highest gel strength was achieved. The irradiation also increased lightness and expressible water amount. Scanning electron micrographs showed that 7 kGy irradiation made the surimi protein gel network more compact. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the degradation of myosin heavy chain (MHC) as the irradiation dose increased, particularly at the 7 and 9 kGy doses. Radiation processing may become a new effective tool in surimi production.

  5. A simple system for staining protein and nucleic acid electrophoresis gels.

    PubMed

    Raymer, Dorian M; Smith, Douglas E

    2007-03-01

    Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels. PMID:17265540

  6. Native Intelligence

    ERIC Educational Resources Information Center

    Seven, Richard

    2006-01-01

    Amid concerns from tribal leaders that No Child Left Behind testing is squeezing out electives that have traditionally covered their history and cultures, an ambitious brace of programs is making Native America part of the core curriculum at David Wolfle Elementary School and other schools in the western Washington State. By tapping into…

  7. Native genomic blotting: high-resolution mapping of DNase I-hypersensitive sites and protein-DNA interactions

    SciTech Connect

    Pauli, U.; Chrysogelos, S.; Stein, J.; Stein, G.

    1988-01-01

    DNase I-hypersensitive sites are observed in the promoter regions of actively expressed genes, potentially active genes, and genes that were once active. The authors have developed an approach that greatly increases the resolution for mapping these sites by electrophoresing genomic DNA on native polyacrylamide gels prior to electroblotting and hybridization. This improved method has been used to scan the promoter and coding region of a cell-cycle-dependent human histone H4 gene with an accuracy of +/- 5-10 base pairs. Protein-DNA interactions can be seen in the autoradiograph as light areas and DNase I-hypersensitive sites as dark bands. Therefore, this method provides a rapid and relatively simple means to accurately localize protein-DNA interactions as well as DNase I-hypersensitive sites, thus directly displaying DNase I hypersensitivity and protein-DNA complexes on one autoradiograph. It also potentially allows the analysis of small changes in DNase I-hypersensitive sites under various biological conditions. With this technique rather large regions of DNA can be screened to determine areas that should be analyzed by more sophisticated methods, such as genomic sequencing or gel retardation assays.

  8. Effect of κ-carrageenan on volume phase transition for polyacrylamide (PAAm) hydrogel using the fluorescence technique

    NASA Astrophysics Data System (ADS)

    Aktaş, Demet Kaya

    2014-03-01

    Steady-state fluorescence (SSF) technique was employed for studying swelling of polyacrylamide (PAAm) gels with various content of κ-carrageenan ( κC). Disc shaped composite hydrogels were prepared by free-radical crosslinking copolymerization of acrylamide (AAm) with various amounts κC. N, N'-methylenebis (acrylamide) (BIS) and ammonium persulfate (APS) were used as crosslinker and initiator, respectively. Pyranine was introduced as a fluorescence probe. Fluorescence intensity of pyranine was monitored during in situ swelling processes of composite gels. It was observed that fluorescence intensity values decreased as swelling is proceeded. Li-Tanaka equation was used to determine the swelling time constants, τ and cooperative diffusion coefficients, D from intensity variations during the swelling processes. It was shown that swelling time constants, τ decreased and diffusion coefficients, D increased as the κC content in the composites are increased.

  9. Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.

    PubMed

    Niyomploy, Ploypat; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Karnchanatat, Aphichart; Sangvanich, Polkit

    2014-03-01

    Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future. PMID:24334192

  10. Effect of Polymer Hydration State on In-Gel Immunoassays.

    PubMed

    Vlassakis, Julea; Herr, Amy E

    2015-11-01

    Applications as diverse as drug delivery and immunoassays require hydrogels to house high concentration macromolecular solutions. Yet, thermodynamic partitioning acts to lower the equilibrium concentration of macromolecules in the hydrogel, as compared to the surrounding liquid phase. For immunoassays that utilize a target antigen immobilized in the hydrogel, partitioning hinders introduction of detection antibody into the gel and, consequently, reduces the in-gel concentration of detection antibody, adversely impacting assay sensitivity. Recently, we developed a single-cell targeted proteomic assay with polyacrylamide gel electrophoresis of single cell lysates followed by an in-gel immunoassay. In the present work, we overcome partitioning that both limits analytical sensitivity and increases consumption of costly detection antibody by performing the immunoassay step after dehydrating the antigen-containing polyacrylamide gel. Gels are rehydrated with a solution of detection antibody. We hypothesized that matching the volume of detection antibody solution with the hydrogel water volume fraction would ensure that, at equilibrium, the detection antibody mass resides in the gel and not in the liquid surrounding the gel. Using this approach, we observe (compared with antibody incubation of hydrated gels): (i) 4-11 fold higher concentration of antibody in the dehydrated gels and in the single-cell assay (ii) higher fluorescence immunoassay signal, with up to 5-fold increases in signal-to-noise-ratio and (iii) reduced detection antibody consumption. We also find that detection antibody signal may be less well-correlated with target protein levels (GFP) using this method, suggesting a trade-off between analytical sensitivity and variation in immunoprobe signal. Our volume-matching approach for introducing macromolecular solutions to hydrogels increases the local in-gel concentration of detection antibody without requiring modification of the hydrogel structure, and thus we anticipate broad applicability to hydrogel-based assays, diagnostics, and drug delivery. PMID:26457450

  11. Native Skies

    NASA Astrophysics Data System (ADS)

    Benningfield, Damond

    2001-03-01

    People native to North America practiced their own version of astronomy. They tracked the motions of the Sun to help them decide when to plant crops, move their camps, and stage sacred rituals. Some tribes built great circles of stones to help them predict the changing seasons. Others built great mounds of earth to reflect the patterns they saw in the heavens and to align their ceremonial centers with the Sun and the Moon.

  12. PREPARATION OF STARCH-G-POLYACRYLAMIDE COPOLYMERS BY REACTIVE EXTRUSION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch and polyacrylamide were prepared by reactive extrusion using a co-rotating twin screw extruder and ammonium persulfate initiator. Feed rates were 109 g/min up to 325 g/min (all components) at a moisture content of 50 percent, with screw speeds in the range 100 rpm to 300 ...

  13. Acrylamide monomer leaching from polyacrylamide-treated irrigation furrows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Water-soluble polyacrylamide (WSPAM), used to reduce erosion in furrow irrigated fields and other agriculture applications, contain less than 0.05% Acrylamide monomer (AMD). The AMD, a potent neurotoxicant and suspected carcinogen, is readily dissolved and transported in flowing water. Deep percol...

  14. Polyacrylamide molecular weight effects on soil infiltration and erosion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seal formation at the surface of smectitic soils during rainstorms reduces soil infiltration rate (IR) and causes runoff and erosion. Surface application of dry anionic polyacrylamide (PAM) with high molecular weight (MW) has been found to be effective in stabilizing soil aggregates, and decreasing ...

  15. HRP-Mediated Synthesis of Starch-Polyacrylamide Graft Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modified starch-based polymers can be engineered for specific properties by combining starch with synthetic polymers through graft copolymerization. Polyacrylamide grafted starches have received a great deal of applications in areas such as superabsorbent paper-making additives, drag reduction and ...

  16. POLYACRYLAMIDE (PAM) IN AGRICULTURE AND ENVIRONMENTAL LAND MANAGEMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anionic polyacrylamide (PAM) has been sold since 1995 to reduce irrigation-induced erosion and enhance infiltration. Its soil stabilizing and flocculating properties improve runoff water quality by reducing sediments, N, dissolved reactive P (DRP) and total P, chemical oxygen demand (COD), pesticid...

  17. PREPARATION OF STARCH-GRAFT-POLYACRYLAMIDE COPOLYMERS BY REACTIVE EXTRUSION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch and polyacrylamide (PAAm) were prepared by reactive extrusion using a co-rotating twin screw extruder and ammonium persulfate initiator. Feed rates were 109 g/min to 325 g/min (all components) at a moisture content of 50%, with screw speeds in the range 100 rpm to 300 rpm...

  18. HRP-Mediated Synthesis of Starch-Polyacrylamide Graft Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modified starch-based polymers can be engineered for specific properties by combining starch with synthetic polymers through graft copolymerization. Polyacrylamide grafted starch have received a great deal of applications in areas such as superabsorbent paper-making additives, drag reduction and te...

  19. 40 CFR 721.10700 - Polyfluorinated alkyl thio polyacrylamide (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10700 Polyfluorinated alkyl thio polyacrylamide (generic). (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substances identified...

  20. POLYACRYLAMIDE EFFECTS ON WATER INFILTRATION IN SANDY LOAM SOILS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some sandy soils of the California San Joaquin Valley have low water infiltration. Electrical conductivity (EC) and sodium adsorption ratio (SAR) of irrigation water greatly affect infiltration rate and hydraulic conductivity of soils. High molecular weight polyacrylamides (PAM) have been shown to i...

  1. Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide

    SciTech Connect

    Kandori, H.; Shichida, Y.; Yoshizawa, T. )

    1989-09-01

    Picosecond laser photolysis of rhodopsin in 15% polyacrylamide gel was performed for estimating absolute absorption spectra of the primary intermediates of cattle rhodopsin (bathorhodopsin and photorhodopsin). Using a rhodopsin digitonin extract embedded in 15% polyacrylamide gel, a precise percentage of bleaching of rhodopsin after excitation of a picosecond laser pulse was measured. Using this value, the absolute absorption spectrum of bathorhodopsin was calculated from the spectral change before and 1 ns after the picosecond laser excitation (corresponding to the difference spectrum between rhodopsin and bathorhodopsin). The absorption spectrum of bathorhodopsin thus obtained displayed a lambda max at 535 nm, which was shorter than that at low temperature (543 nm) and a half band-width broader than that measured at low temperature. The oscillator strength of bathorhodopsin at room temperature was smaller than that at low temperature. The absolute absorption spectrum of photorhodopsin was also estimated from the difference spectrum measured at 15 ps after the excitation of rhodopsin, assuming a sequential conversion of photorhodopsin to bathorhodopsin. Its lambda max was located at approximately 570 nm, and the oscillator strength was smaller than those of rhodopsin and bathorhodopsin.

  2. Hydrophobicity-induced prestaining for protein detection in polyacrylamide gel electrophoresis.

    PubMed

    Li, Zhe; Guan, Weijiang; Lu, Chao; Zhou, Xi-Rui; Luo, Shi-Zhong; You, Ying; Ouyang, Jin

    2016-02-01

    An AIE fluorescent surfactant has been first used to prestain protein by ultrastrong hydrophobic interaction between fluorescent surfactants and proteins, distinguishing from the most widely used poststaining strategies by employing AIE molecules with weak hydrophobic characteristics. A mixture of proteins with variable molecular weights has been detected. PMID:26771025

  3. Ultrasensitive Stain for Proteins in Polyacrylamide Gels Shows Regional Variation in Cerebrospinal Fluid Proteins

    NASA Astrophysics Data System (ADS)

    Merril, Carl R.; Goldman, David; Sedman, Sylvia A.; Ebert, Michael H.

    1981-03-01

    A new silver stain for electrophoretically separated polypeptides can be rapidly and easily used and can detect as little as 0.01 nanogram of protein per square millimeter. When employed with two-dimensional electrophoresis, it should permit qualitative and quantitative characterization of protein distributions in body fluids and tissues. It has been used to demonstrate regional variations in cerebrospinal fluid proteins.

  4. Application of multiple levels of fluid shear stress to endothelial cells plated on polyacrylamide gels

    PubMed Central

    Galie, P. A.; van Oosten, A.; Chen, C. S.

    2015-01-01

    Measurements of endothelial cell response to fluid shear stress have previously been performed on unphysiologically rigid substrates. We describe the design and implementation of a microfluidic device that applies discrete levels of shear stress to cells plated on hydrogel-based substrates of physiologicallyrelevant stiffness. The setup allows for measurements of cell morphology and inflammatory response to the combined stimuli, and identifies mechanisms by which vascular stiffening leads to pathological responses to blood flow. We found that the magnitude of shear stress required to affect endothelial cell morphology and inflammatory response depended on substrate stiffness. Endothelial cells on 100 Pa substrates demonstrate a greater increase in cell area and cortical stiffness and decrease in NF-?B nuclear translocation in response to TNF-? treatment compared to controls than cells plated on 10 kPa substrates. The response of endothelial cells on soft substrates to shear stress depends on the presence of hyaluronan (HA). These results emphasize the importance of substrate stiffness on endothelial function, and elucidate a means by which vascular stiffening in aging and disease can impact the endothelium. PMID:25573790

  5. A LOW-COST HIGH THROUGHPUT POLYACRYLAMIDE GEL ELECTROPHORESIS SYSTEM FOR GENOTYPING WITH MICROSATELLITE DNA MARKERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite DNA markers are widely used in genetic research but the genotyping cost with this marker system is high and the throughput is limited with conventional methods. The objective of this paper is to introduce a low-cost, high-throughput system developed in our laboratories for the detect...

  6. Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes.

    PubMed

    Ganesh, G; Kumar, T K; Pandian, S T; Yu, C

    2000-11-20

    The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes. PMID:11086192

  7. Identification of peanut hybrids using microsatellite markers and horizontal polyacrylamide gel electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In peanut hybridization, it is very important to be able to distinguish selfs from true hybrids to save time and resources. To help facilitate this effort and provide molecular distinction between selfs from hybrids, DNA was extracted from leaf tissue of F1 or F2 plants, and SSR markers were amplif...

  8. Resistance of structure and antigenic determinations of native hexon of type 1 adenovirus to protease

    SciTech Connect

    Kiseleva, E.K.; Khil'ko, S.N.; Grigor'ev, V.G.; Dyachenko, N.S.; Vantsak, N.P.; Tikhonenko, T.I.

    1987-02-10

    Native hexon capsomers (trimers) of human type 1 adenovirus (Ad hl) were labeled with /sup 125/I and subjected to cleavage by trypsin, chymotrypsin, and papain. The hydrolysis of the hexon of Ad 1 by these enzymes is limited and in each case a set of relatively large fragments, which are not cleaved during prolonged hydrolysis, is formed. The degree of hydrolysis of the Ad hl hexon increases in the order trypsin < chymotrypsin < papain, the molecular weight of the largest fragments constituting 80,000, 40,000, and 32,000, respectively. At a decreased temperature all the large fragments of the hydrolysates obtained are confined in aggregates (the cores of the hexon), similar in structure to the original hexon trimers, the papain core of the Ad hl hexon being the most stable during electrophoresis in polyacrylamide gel and the chymotrypsin core being the least. A radioimmunoprecipitation analysis showed that denatured fragments of the tryptic, chymotryptic, and papain hydrolysates do not possess antigenic activity.

  9. Isolation and Characterization of a Native Cell Wall Complex from Neisseria meningitidis

    PubMed Central

    Zollinger, W. D.; Kasper, D. L.; Veltri, B. J.; Artenstein, M. S.

    1972-01-01

    A cell wall complex has been isolated by gentle methods from both the medium supernatant fluid and whole organisms of Neissieria meningitidis cultures. The two types of preparations have been shown to be essentially identical on the basis of chemical composition, electron microscopy, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Four major components were identified in the complex: group-specific polysaccharide (4 to 10%), protein (45 to 65%), lipopolysaccharide (10 to 25%), and lipid (15 to 30%). The whole complex was found to be immunogenic in rabbits and to elicit production of antibody directed against the protein, the group-specific polysaccharide, and the lipopolysaccharide components. The isolated protein component was also found to be immunogenic in rabbits and to elicit production of serotype-specific antibody. The protein component was found to produce a band pattern in SDS-PAGE that is simple, reproducible, and strain dependent. The lipopolysaccharide component was found to have chemical and biological properties characteristic of bacterial endotoxin. We propose that this complex is representative of the outer trilaminar membrane of the meningococcal cell envelope in its native state. Images PMID:4629210

  10. 2D gel proteomics: an approach to study age-related differences in protein abundance or isoform complexity in biological samples.

    PubMed

    Kim, Helen; Eliuk, Shannon; Deshane, Jessy; Meleth, Sreelatha; Sanderson, Todd; Pinner, Anita; Robinson, Gloria; Wilson, Landon; Kirk, Marion; Barnes, Stephen

    2007-01-01

    This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat. PMID:17634592

  11. Immobilization of enzymes on alginic acid-polyacrylamide copolymers

    SciTech Connect

    Kumaraswamy, M.D.K.; Panduranga R.K.; Thomas J.K.; Santappa, M.

    1981-08-01

    In this report, the authors present initial results and limitations of a polymeric system for the immobilization of enzymes. Enzymes attached to insoluble polymers of natural and synthetic origin are gaining importance in many industrial and biomedical applications. Graft copolymers are used as enzyme supports and in this study a novel polymeric system of alginic acid-polyacrylamide graft copolymer is described which was used for immobilizing enzymes. (Refs. 4).

  12. Aerosol gels

    NASA Technical Reports Server (NTRS)

    Sorensen, Christopher M. (Inventor); Chakrabarti, Amitabha (Inventor); Dhaubhadel, Rajan (Inventor); Gerving, Corey (Inventor)

    2010-01-01

    An improved process for the production of ultralow density, high specific surface area gel products is provided which comprises providing, in an enclosed chamber, a mixture made up of small particles of material suspended in gas; the particles are then caused to aggregate in the chamber to form ramified fractal aggregate gels. The particles should have a radius (a) of up to about 50 nm and the aerosol should have a volume fraction (f.sub.v) of at least 10.sup.-4. In preferred practice, the mixture is created by a spark-induced explosion of a precursor material (e.g., a hydrocarbon) and oxygen within the chamber. New compositions of matter are disclosed having densities below 3.0 mg/cc.

  13. Protein electrophoresis in agarose gels for separating high molecular weight proteins.

    PubMed

    Greaser, Marion L; Warren, Chad M

    2012-01-01

    Very large proteins (subunit sizes >200 kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4,000 kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer has been found to be a key technical procedure for obtaining optimum resolution. PMID:22585481

  14. SEPARATION AND IDENTIFICATION OF SOYBEAN LEAF PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted las...

  15. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissecte...

  16. Dielectric properties of gel collected from shark electrosensors

    NASA Astrophysics Data System (ADS)

    Hughes, Mary E.; Brown, Brandon R.; Hutchison, John C.; Murray, Royce W.

    2003-03-01

    To investigate the physical mechanism of the electric sense, we present an initial characterization of the dielectric properties of the glycoprotein gel that fills the electrosensitive organs of marine elasmobranches (sharks, skates, and rays). To ascertain the properties of the gel, low-frequency impedance spectroscopy is used. The impedance data collected from a dialyzed sample show large values of static permittivity and a loss peak corresponding to a long relaxation time (about 1 ms). Impedance measurements of the native (nondialyzed) gel reliable to 0.1 Hz will be presented and compared to the dialyzed gel. Ramifications of the gel's dielectric properties for the electric sense will be explored.

  17. Polyelectrolyte gels in electric fields: a theoretical and experimental approach

    NASA Astrophysics Data System (ADS)

    Guelch, Rainer W.; Holdenried, Jens; Weible, Andrea; Wallmersperger, Thomas; Kroeplin, Bernd

    2000-06-01

    In order to gain more insight into basic principles of the nature of polymer hydrogels which are able to execute work by large deformations in electric fields, this study is mainly focused on those gels with a polyacrylamide backbone being very suitable for considerably varying their physicochemical properties simply by specific copolymerization. In the experimental part, the Donnan potential has been registered for the first time in PAAm/PAA gels in varying electric fields and different chemical environments with a new microelectrode technique. The mechanical properties of the gels have been characterized by measurements of swelling ratio, elastic modulus and being in dynamics under various stimuli. In the theoretical part, a model based both on this theory and the measured mechanical parameters, the bending dynamics of a polyelectrolyte gel in an electric field can be evaluated. Numerical simulations employing finite element discretization demonstrate the potential and the validity of the model. A promising correlation between theory and experiment could be shown.

  18. Measurements of elastic moduli of silicone gel substrates with a microfluidic device.

    PubMed

    Gutierrez, Edgar; Groisman, Alex

    2011-01-01

    Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments. PMID:21980487

  19. Improvement of Catalytic Efficiency, Thermo-stability and Dye Decolorization Capability of Pleurotus ostreatus IBL-02 laccase by Hydrophobic Sol Gel Entrapment

    PubMed Central

    2012-01-01

    Background In serious consideration of the worldwide environmental issues associated with the extensive use of the textile dyes and effluents generated thereof, the scientists across the world are in search for potential treatment technologies for their treatment. In such scenario the ligninolytic enzymes provide a potential alternative because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents. Results Laccase produced from Pleurotus ostreatus IBL-02 during decolorization of the reactive textile dye Drimarene brilliant red K-4BL (DBR K-4BL) was purified and immobilized by hydrophobic gel entrapment. The crude laccase was 4.2-fold purified with specific activity of 573.52 U/mg after passing through the DEAE-Sepharose ion exchange and Sephadex-G-100 chromatography columns. P. ostreatus IBL-02 laccase was found to be a homogenous monomeric protein as evident by single band corresponding to 67 kDa on native and sodium dodesylsulfate polyacrylamide gel electrophoresis (PAGE). The laccase was immobilized by entrapment in Sol–gel matrix of trimethoxysilane (T) and proplytetramethoxysilane (P) prepared using different T:P molar ratios. The free and immobilized laccases were compared to investigate the effect of immobilization on catalytic efficiency and thermo-stability features. Laccase immobilized in the Sol–gel of 1:5 T:P ratio was optimally active and thermo-stable fraction at pH 5, 60°C with half-life of 3 h and 50 min. Laccases immobilized in 1:2 and 1:5 T:P ratio gels had significantly higher Km (83 and100mM) and Vmax (1000 and 1111 mM/mg) values as compared to free laccase. After 5 h reaction time varying decolorization percentages with a maximum of 100% were achieved for different dyes and effluents. Conclusions In summary, P. ostreatus IBL-02 laccase was immobilized by entrapping in a Sol–gel matrix with an objective to enhance its catalytic and stability properties. Sol–gel entrapped laccase presented potential efficiency as a biocatalyst when applied for decolorization of different dyes and effluents. The main benefits of the Sol–gel matrix immobilization processes are the eco-friendly approach, chemical free and energy saving reaction conditions. PMID:23021344

  20. Zeta Potential Measurements of Glyoxalated Polyacrylamide (GPAM) Resins

    NASA Astrophysics Data System (ADS)

    Libi, Sumit; Shrestha, Apsana; Norwood, David; Boone, Steven

    2013-03-01

    We will describe the use of a NICOMP 380 ZLS light scattering instrument (Particle Sizing Systems) to measure the zeta potential of glyoxalated polyacrylamide (GPAM) resins used in the paper industry. These experiments are part of a broader study of GPAM molecule properties (molecular weight, RMS radius, contour and persistence length) intended to understand differences in performance between various GPAM resins (specifically, differences in drainage performance during paper processing and wet/dry strength of paper). Additionally, zeta potential measurements help to understand the long term stability of these resins. Data and results obtained from the experiment will be presented.

  1. Preparation and characterization of polyacrylamide-silver nanocomposites

    NASA Astrophysics Data System (ADS)

    Yin, Yadong; Xu, Xiangling; Ge, Xuewu; Zhang, Zhicheng

    1998-11-01

    Polyacrylamide-silver nanocomposites are successfully prepared by irradiating the aqueous solution of AgNO 3 and acrylamide monomer with 60Co γ-ray. The composites are found to contain nanometer silver particles with a narrow size distribution and a homogeneous dispersion. The existing of isopropanol (as a hydroxyl radical scavenger and chain transfer agent) in system affects the properties of both the dispersed phase and matrix of the nanocomposites. The fast-formed polymer chains probably play a key role in preventing the aggregation of silver particles which are reduced later.

  2. Effects of polymer concentration on the morphology of calcium phosphate crystals formed in polyacrylamide hydrogels

    NASA Astrophysics Data System (ADS)

    Yokoi, Taishi; Kawashita, Masakazu; Ohtsuki, Chikara

    2013-11-01

    Growing crystals in hydrogels is an attractive method to form inorganic solids with designed morphology under ambient conditions. Precipitation of the inorganic solids in a hydrogel matrix can be regarded as mimicking the process of biomineralization. In the construction of biominerals, an organic template composed of insoluble macromolecules is used to control the crystal growth of the inorganic compounds. The morphological control in biomineralization can be applied to artificial reaction systems. In this study, the morphology of calcium phosphate crystals formed in polymeric hydrogels of various polymer concentrations was investigated. Spherical octacalcium phosphate (OCP) precipitated in the polyacrylamide (PAAm) hydrogels. Fibrous crystals gradually covered the surface of the spherical crystals as the polymer concentration of the gel increased. The morphology of the OCP crystals changed from sea urchin shapes to wool-ball shapes with increasing PAAm concentration. The morphological change is generated by the template effect of the polymer wall, which is made up of stacked PAAm sheets, surrounding the spherical OCP crystals.

  3. Isolation and Characterization of Polyacrylamide-Degrading Bacteria from Dewatered Sludge

    PubMed Central

    Yu, Feng; Fu, Ruimin; Xie, Yun; Chen, Wuling

    2015-01-01

    Polyacrylamide (PAM) is a water-soluble polymer that is widely used as a flocculant in sewage treatment. The accumulation of PAM affects the formation of dewatered sludge and potentially produces hazardous monomers. In the present study, the bacterial strain HI47 was isolated from dewatered sludge. This strain could metabolize PAM as its sole nutrient source and was subsequently identified as Pseudomonas putida. The efficiency of PAM degradation was 31.1% in 7 days and exceeded 45% under optimum culture condition (pH 7.2, 39 °C and 100 rpm). The addition of yeast extract and glucose improved the bacterial growth and PAM degradation. The degraded PAM samples were analyzed by gel-filtration chromatography, Fourier transform infrared and high-performance liquid chromatography. The results showed that high-molecular-weight PAM was partly cleaved to small molecular oligomer derivatives and part of the amide groups of PAM had been converted to carboxyl groups. The biodegradation did not accumulate acrylamide monomers. Based on the SDS-PAGE and N-terminal sequencing results, the PAM amide groups were converted into carboxyl groups by a PAM-induced extracellular enzyme from the aliphatic amidase family. PMID:25893998

  4. A convenient green preparation of layered double hydroxide/polyacrylamide nanocomposite hydrogels with ultrahigh deformability.

    PubMed

    Wu, Lianying; Hu, Ziqiao; Chen, Guangming; Li, Zhibo

    2015-12-14

    A convenient green preparation method has been developed to achieve layered double hydroxide (LDH)/polymer nanocomposite (NC) hydrogels. In contrast to previous publications using toxic organic solvent of formamide or methanol in LDH exfoliation or anion exchange, the interlayer anion exchange and exfoliation of LDH are completed in one step with the help of an amino acid (L-serine). The LDH/polyacrylamide (PAM) NC hydrogels are achieved by a convenient exfoliation-adsorption in situ polymerization method. The exfoliation of LDH is characterized by dynamic light scattering and transmission electron microscopy. Interestingly, the developed NC hydrogels reveal ultrahigh deformability and extraordinary stretchability, confirmed by qualitative images and qualitative tensile and compression tests. The molecular mechanism for the ultrahigh deformability and extraordinary stretchability is discussed by crosslinking density, inter-crosslinking molecular weight and swelling tests. We believe that the findings reported herein will deepen our understanding towards the chemistry of network soft materials including gels, and further widen the applications of polymer hydrogels in mechanical devices such as artificial muscles, biomedical devices and drug delivery systems. PMID:26412191

  5. 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.

  6. Electric Field Enhanced Diffusion of Salicylic Acid through Polyacrylamide Hydrogels

    NASA Astrophysics Data System (ADS)

    Niamlang, Sumonman; Sirivat, Anuvat

    2008-03-01

    The release mechanisms and the diffusion coefficients of salicylic acid -loaded polyacrylamide hydrogels were investigated experimentally by using a modified Franz-diffusion cell at 37 ^oC to determine the effects of crosslinking ratio and electric field strength. A significant amount of salicylic acid is released within 48 hours from the hydrogels of various crosslinking ratios, with and without electric field. The release characteristic follows the Q vs. t^1/2 linear relationship. Diffusion coefficient initially increases with increasing electric field strength and reaches the maximum value at electric field strength of 0.1 V; beyond that it decreases with electric field strength and becomes saturated at electric field strength of 5 V. The diffusion coefficient increases at low electric field strength (less 0.1 V) as a result of the electrophoresis of the salicylic acid, the expansion of pore size, and the induced pathway in pigskin. For electric field strength higher than 0.1 V, the decrease in the diffusion coefficient is due to the reduction of the polyacrylamide pore size. The diffusion coefficient obeys the scaling behavior D/Do=(drug size/pore size)^m, with the scaling exponent m equal to 0.93 and 0.42 at electric fields of 0 and 0.1 V, respectively.

  7. Native American Discursive Tactic

    ERIC Educational Resources Information Center

    Black, Jason Edward

    2013-01-01

    This essay derives from a course called ‘"The Rhetoric of Native America,’" which is a historical-critical survey of Native American primary texts. The course examines the rhetoric employed by Natives to enact social change and to build community in the face of exigencies. The main goal of exploring a native text (particularly, Simon Pokagon's…

  8. Biohybrid Branched Copolymers of Controlled Nanostructure Based on Polyacrylamide Grafted to Dextran or Dextran Sulphate Backbone

    NASA Astrophysics Data System (ADS)

    Kutsevol, N.; Soushko, R.; Guenet, J.-M.; Melnyk, N.

    2008-08-01

    Branched polymers Dextran-graft-Polyacrylamide and Dextran Sulphate-graft-Polyacrylamide copolymers with long polysaccharide backbone and various amount and length of PAA-grafts were synthesised by radical polymerization using Ce(IV)/HNO3 redox system. It was established, that these copolymers have expanded conformation in solution and are able to intramolecular structure formation. Grafting efficiency is conditioned by amount of initiator and Dextran (or Dextran Sulphate) conformation in solution. It was shown that increase in content of copolymer Polyacrylamide component in Dextran Sulphate-graft-Polyacrylamide copolymers causes decrease in size of macromolecule due to blocking of polyion charge of main polysaccharide chain by counterions.

  9. Gel protein capillary extraction apparatus. electronic protein transfer.

    PubMed

    Cooper, Jonathan W; Gao, Jun; Lee, Cheng S

    2002-03-01

    A gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to a fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field amplification and sample stacking during the extraction process. Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and beta-galactosidase (116 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 50 ng. The SDS-cytochrome c complexes extracted from a 50-ng protein loading are concentrated in a 30-nL solution plug inside the capillary with an estimated concentration of 0. 1 mg/mL or 10(-5) M. The capillary format allows the straightforward integration of a miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification. PMID:11924982

  10. Low-velocity super-lubrication of sodium-alginate/polyacrylamide ionic-covalent hybrid double-network hydrogels.

    PubMed

    Li, Xuefeng; Wu, Chu; Yang, Qian; Long, Shijun; Wu, Chonggang

    2015-04-21

    Structural and frictional behaviours of sodium alginate (SA)/polyacrylamide (PAAm) ionic-covalent hybrid, sequential double-network (DN) hydrogels against glass have been investigated in water, NaCl and CaCl2 aqueous solutions using a rotational rheometer. Dilution of adsorptive elastohydrodynamic friction for the PAAm covalent network with repulsive hydrodynamic lubrication for the minor SA ionic network was found to control the frictional stresses of the SA/PAAm gels within between those of the SA and PAAm single-network gels. A tentative qualitative model was proposed to describe the impact of ionic environmental solution on the frictional behaviour of the hybrid gel by selectively affecting the SA-network structure and friction. It was revealed that strong Debye shielding in the NaCl solution significantly reduced the thickness of the electric double layer for hydrodynamic lubrication of the SA network, which made the SA/PAAm gel's friction the highest among the three solutions. Dramatically increased ionic cross-linking of the SA network in the CaCl2 solution, although effectively mediated by the PAAm-network flexible skeleton, still functioned partially to conserve a portion of the SA fractional boundary-friction at the interface, making the friction of the hybrid gel intermediate among the three solutions. In contrast, extreme hydration of the SA network in water sharply increased the volume fraction of its unshielded hydrodynamic lubrication at the interface, which greatly reduced the SA/PAAm's friction to the lowest among the three solutions. We have thus incorporated for the first time both super-lubrication (frictional coefficients of below 10(-2) over low sliding-velocities of 3 × 10(-5) to 2 × 10(-3) m s(-1)) and previously reported high fracture energy (over 9000 J m(-2)) into a single ionic-covalent hybrid DN hydrogel, which is the SA/PAAm (∼1/8.5 w/w) gel in water. Effects of inversion of DN-formation sequence further indicated that frictional behaviours (i.e. frictional stress-sliding velocity profiles) of the hybrid sequential DN hydrogels (SA/PAAm and PAAm/SA), respectively, were primarily determined by those of the second networks (PAAm and SA), presumably due to the formation of first-second network "core-shell" structures at the blob scale. Frictional stress of the SA/PAAm gel was increased monotonically with external normal pressure at all of the sliding velocities investigated in the three solutions, which was in agreement with the predictions from the repulsion-adsorption model proposed by Gong et al. PMID:25735912

  11. Neutron insensitive depolymerization of polyacrylamide for californium-252 photon dosimetry.

    PubMed

    Moyers, M F; Beach, J L

    1982-01-01

    A photon dosimeter based on degradation of polyacrylamide (PAA) in dilute aqueous solution has been found to be insensitive to neutrons from Cf-252, independent of photon energy from 34 keVeff to 1.33 MeV, and useful over a dose range of 1 to 100 Gy. Because this dilute polymer solution is virtually 100% water, it is nearly tissue equivalent for photon absorption as well as neutron scatter and attenuation. Radiation response was determined by measuring decreases in viscosity three days after irradiation. The measured response is dose rate independent from 10 to over 300 Gy/h but increases rapidly at lower dose rates. Long-term, radiation-induced viscosity changes were also observed. The many advantages of this tissue equivalent system for photon/neutron dosimetry suggest the need for further studies of the observed over-response at low dose rates. PMID:7155069

  12. Hydrolyzed polyacrylamide biodegradation and mechanism in sequencing batch biofilm reactor.

    PubMed

    Yan, Miao; Zhao, Lanmei; Bao, Mutai; Lu, Jinren

    2016-05-01

    An investigation was performed to study the performance of a sequencing batch biofilm reactor (SBBR) to treat hydrolyzed polyacrylamides (HPAMs) and to determine the mechanisms of HPAM biodegradation. The mechanisms for the optimized parameters that significantly improved the degradation efficiency of the HPAMs were investigated by a synergistic effect of the co-metabolism in the sludge and the enzyme activities. The HPAM and TOC removal ratio reached 54.69% and 70.14%. A significant decrease in the total nitrogen concentration was measured. The carbon backbone of the HPAMs could be degraded after the separation of the amide group according to the data analysis. The HPLC results indicated that the HPAMs could be converted to polymer fragments without the generation of the acrylamide monomer intermediate. The results from high-throughput sequencing analysis revealed proteobacterias, bacteroidetes and planctomycetes were the key microorganisms involved in the degradation. PMID:26896716

  13. High-resolution gel dosimetry of a HDR brachytherapy source using normoxic polymer gel dosimeters: Preliminary study

    NASA Astrophysics Data System (ADS)

    Hurley, C.; McLucas, C.; Pedrazzini, G.; Baldock, C.

    2006-09-01

    Polymer gel dosimetry has been shown to be an effective tool in the analysis of radiotherapy treatments in cancer therapy, being used to map the dose distribution around an irradiation pattern of a polymer gel dosimeter. Combined with high-resolution magnetic resonance imaging (MRI), polymer gel dosimetry can be an effective dosimetry tool to map dose distributions with high spatial resolution (˜100 μm). Previously polyacrylamide polymer gel dosimetry required a strict hypoxic environment to protect the gel from oxygen infiltration as oxygen inhibits the polymerization reaction used to correlate to absorbed dose. However, with the advent of normoxic polymer gels, a strict hypoxic environment is not required. Normoxic polymer gel dosimeters can be manufactured under normal atmospheric conditions. This study assessed the use of a MAGIC normoxic polymer gel dosimeter to accurately map the dose distribution of a single-line irradiation and a point source irradiation from a brachytherapy radiation source administered through a nylon catheter inserted into the gel dosimeter. The phantoms were irradiated to a dose of 10 Gy at 2 mm from the source center and imaged using high-resolution MRI with an in-plane pixel size of 0.1055 mm/pixel. Good agreement was found between the dose points predicted by the computer treatment-planning system and the measured normalized dose profiles in the gel dosimeter. The use of normoxic polymer gel dosimeters with high-resolution MRI evaluation shows promise as an effective tool in applications requiring accurate dose distributions in high resolution, such as intravascular brachytherapy.

  14. 21 CFR 872.3480 - Polyacrylamide polymer (modified cationic) denture adhesive.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... adhesive. 872.3480 Section 872.3480 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... polymer (modified cationic) denture adhesive. (a) Identification. A polyacrylamide polymer (modified cationic) denture adhesive is a device composed of polyacrylamide polymer (modified cationic) intended...

  15. 21 CFR 872.3480 - Polyacrylamide polymer (modified cationic) denture adhesive.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... adhesive. 872.3480 Section 872.3480 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... polymer (modified cationic) denture adhesive. (a) Identification. A polyacrylamide polymer (modified cationic) denture adhesive is a device composed of polyacrylamide polymer (modified cationic) intended...

  16. 21 CFR 872.3480 - Polyacrylamide polymer (modified cationic) denture adhesive.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... adhesive. 872.3480 Section 872.3480 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... polymer (modified cationic) denture adhesive. (a) Identification. A polyacrylamide polymer (modified cationic) denture adhesive is a device composed of polyacrylamide polymer (modified cationic) intended...

  17. 21 CFR 872.3480 - Polyacrylamide polymer (modified cationic) denture adhesive.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... adhesive. 872.3480 Section 872.3480 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... polymer (modified cationic) denture adhesive. (a) Identification. A polyacrylamide polymer (modified cationic) denture adhesive is a device composed of polyacrylamide polymer (modified cationic) intended...

  18. 21 CFR 872.3480 - Polyacrylamide polymer (modified cationic) denture adhesive.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... adhesive. 872.3480 Section 872.3480 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... polymer (modified cationic) denture adhesive. (a) Identification. A polyacrylamide polymer (modified cationic) denture adhesive is a device composed of polyacrylamide polymer (modified cationic) intended...

  19. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.

    PubMed

    Rosenfeld, J; Capdevielle, J; Guillemot, J C; Ferrara, P

    1992-05-15

    We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis. PMID:1524213

  20. Chemical gel barriers as low-cost alternative to containment and in situ cleanup of hazardous wastes to protect groundwater

    SciTech Connect

    1997-01-01

    Chemical gel barriers are being considered as a low-cost alternative for containment and in situ cleanup of hazardous wastes to protect groundwater. Most of the available gels in petroleum application are non-reactive and relative impermeable, providing a physical barriers for all fluids and contaminants. However, other potential systems can be envisioned. These systems could include gels that are chemically reactive and impermeable such that most phase are captured by the barriers but the contaminants could diffuse through the barriers. Another system that is chemically reactive and permeable could have potential applications in selectivity capturing contaminants while allowing water to pass through the barriers. This study focused on chemically reactive and permeable gel barriers. The gels used in experiment are DuPont LUDOX SM colloidal silica gel and Pfizer FLOPAAM 1330S hydrolyzed polyacrylamide (HPAM) gel.

  1. Experiencing Native American Culture.

    ERIC Educational Resources Information Center

    Darou, Wes G.

    1980-01-01

    Presents four experiential excercises based on Native American culture for use in education, communication training, and counseling. These excercises are intended as vehicles for personal growth and aids to learning about Native American culture. (RC)

  2. Native Health Research Database

    MedlinePlus

    ... Havasupai Hawaiian Native Ho-Chunk Hoh Hoopa Hooper Bay, Native Village of Hopi Houma Hualapai Hupa Innu ... of Oklahoma Seminole Tribe of Florida Seneca Shoalwater Bay Shoshone Shoshone-Bannock Shuara Siberian Chukotka Siletz Sioux ...

  3. How deeply cells feel: methods for thin gels

    NASA Astrophysics Data System (ADS)

    Buxboim, Amnon; Rajagopal, Karthikan; Brown, Andre'E. X.; Discher, Dennis E.

    2010-05-01

    Tissue cells lack the ability to see or hear but have evolved mechanisms to feel into their surroundings and sense a collective stiffness. A cell can even sense the effective stiffness of rigid objects that are not in direct cellular contactlike the proverbial princess who feels a pea placed beneath soft mattresses. How deeply a cell feels into a matrix can be measured by assessing cell responses on a controlled series of thin and elastic gels that are affixed to a rigid substrate. Gel elasticity E is readily varied with polymer concentrations of now-standard polyacrylamide hydrogels, but to eliminate wrinkling and detachment of thin gels from an underlying glass coverslip, vinyl groups are bonded to the glass before polymerization. Gel thickness is nominally specified using micron-scale beads that act as spacers, but gels swell after polymerization as measured by z-section, confocal microscopy of fluorescent gels. Atomic force microscopy is used to measure E at gel surfaces, employing stresses and strains that are typically generated by cells and yielding values for E that span a broad range of tissue microenvironments. To illustrate cell sensitivities to a series of thin-to-thick gels, the adhesive spreading of mesenchymal stem cells was measured on gel mimics of a very soft tissue (e.g. brain, E ~ 1 kPa). Initial results show that cells increasingly respond to the rigidity of an underlying 'hidden' surface starting at about 10-20 m gel thickness with a characteristic tactile length of less than about 5 m.

  4. Rebuilding Native American communities.

    PubMed

    Coyhis, Don; Simonelli, Richard

    2005-01-01

    The Wellbriety Movement in Native American communities draws on the wisdom and participation of traditional elders. Beginning with a basic community teaching called the Four Laws of Change and the Healing Forest Model, the Wellbriety Movement blends Medicine Wheel knowledge with the 12 Steps of Alcoholics Anonymous to provide culture-specific addiction recovery help for Native Americans. The four Journeys of the Sacred Hoop brought the Wellbriety Movement to Native and non-Native people alike from 1999 to 2003. PMID:15828416

  5. Alaska Natives & the Land.

    ERIC Educational Resources Information Center

    Arnold, Robert D.; And Others

    Pursuant to the Native land claims within Alaska, this compilation of background data and interpretive materials relevant to a fair resolution of the Alaska Native problem seeks to record data and information on the Native peoples; the land and resources of Alaska and their uses by the people in the past and present; land ownership; and future…

  6. Native American Healing Traditions

    ERIC Educational Resources Information Center

    Portman, Tarrell A. A.; Garrett, Michael T.

    2006-01-01

    Indigenous healing practices among Native Americans have been documented in the United States since colonisation. Cultural encapsulation has deterred the acknowledgement of Native American medicinal practices as a precursor to folk medicine and many herbal remedies, which have greatly influenced modern medicine. Understanding Native American

  7. Native American Healing Traditions

    ERIC Educational Resources Information Center

    Portman, Tarrell A. A.; Garrett, Michael T.

    2006-01-01

    Indigenous healing practices among Native Americans have been documented in the United States since colonisation. Cultural encapsulation has deterred the acknowledgement of Native American medicinal practices as a precursor to folk medicine and many herbal remedies, which have greatly influenced modern medicine. Understanding Native American…

  8. Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40.

    PubMed

    Kim, Sung Chan; Kang, Seung Ha; Choi, Eun Young; Hong, Yeon Hee; Bok, Jin Duck; Kim, Jae Yeong; Lee, Sang Suk; Choi, Yun Jaie; Choi, In Soon; Cho, Kwang Keun

    2016-01-01

    A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine-Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively. PMID:26732336

  9. Cloning and Characterization of an Endoglucanase Gene from Actinomyces sp. Korean Native Goat 40

    PubMed Central

    Kim, Sung Chan; Kang, Seung Ha; Choi, Eun Young; Hong, Yeon Hee; Bok, Jin Duck; Kim, Jae Yeong; Lee, Sang Suk; Choi, Yun Jaie; Choi, In Soon; Cho, Kwang Keun

    2016-01-01

    A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine–Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively. PMID:26732336

  10. Characterization of Helicoverpa armigera gut proteinases and their interaction with proteinase inhibitors using gel X-ray film contact print technique.

    PubMed

    Harsulkar, A M; Giri, A P; Gupta, V S; Sainani, M N; Deshpande, V V; Patankar, A G; Ranjekar, P K

    1998-06-01

    Since Helicoverpa armigera is a devastating pest, an attempt was made to separate its gut proteinases and assess their diversity. Gelatin coating present on the X-ray film was used as a substrate to detect electrophoretically separated proteinases of H. armigera gut extract on native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and isoelectric focusing gels. The method involves electrophoresis, followed by washing the gel with Triton X-100 in case of SDS-PAGE, equilibration of the gel in proteinase assay buffers, overlaying the gel on X-ray film followed by washing the film with hot water to remove hydrolyzed gelatin revealing bands of proteinase activity. Using this protocol, at least six different proteinase isoforms were detected in H. armigera gut contents while three isoproteinases were identified in a commercial bacterial proteinase preparation. Adoption of the technique facilitated characterization of the H. armigera gut proteinases (HGP) and provided an easy tool to study the properties of the individual proteinases without purification. The approximate molecular masses of HGP as determined by SDS-PAGE were: 172.9, 59.3, 54.9, 47.6, 44.1 and 41.6 kDa, and of bacterial proteinases: 180.7, 127.3 and 95.3 kDa. The isoelectric point (pI) values of HGP and bacterial proteinase were in the range of 5.1-7.1 and 3.5-7.7, respectively. Some of the HGP isoforms were found to be highly pH-sensitive and showed activity only at pH 10.0. The major HGPs were inhibited by phenylmethylsulfonyl fluoride but not by (4-amidinophenyl)-methanesulfonyl fluoride. Incubation of HGP-resolved electrophoretic gel strips in chickpea or winged bean proteinase inhibitor solution permitted identification of specific inhibitors of individual proteinases and revealed that the major HGPs were insensitive to chickpea inhibitors whereas winged bean inhibitors effectively inhibited all the HGPs. Our results suggest that considerable variability exists among the isoproteinases of H. armigera gut with respect to their pH optima and sensitivity towards chemical and plant proteinase inhibitors. Such diversity is of immense biological significance as it explains the polyphagous nature of the insect which imparts unique adaptability to it against the defensive proteinase inhibitors of its wide range of host plants. PMID:9694289

  11. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    PubMed Central

    Kaer, Kristel; Mätlik, Kert; Metsis, Madis; Speek, Mart

    2008-01-01

    Background In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence. Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc), separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (p75NTR) gene. Conclusion The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis. PMID:18533036

  12. Quantitation and characterization of rat tissue metallothioneins by gel electrophoresis

    SciTech Connect

    Lin, L.Y.; McCormick, C.C.

    1986-03-05

    A discontinuous gradient gel electrophoretic system was developed to quantitate and characterize metallothionein (MT) in rat tissue. Vertical slab separating gels (1.5 mm x 14 cm x 12 cm) consisted of a linear polyacrylamide gradient 7.5 to 30% T and 5% Bis. The stacking gels (3% T and 20% Bis) were photopolymerized using riboflavin as the catalyst. Liver cytosols were prepared from rats which received (i.p.) various amounts of Zn (5 mg/kg BW) or Cd (2.5 mg/kg BW). Purified MT was prepared by gel filtration and DEAE ion-exchange chromatography. Cytosols were heated (80/sup 0/C, 2 min) and centrifuged to obtain a supernatant. An appropriate amount of supernatant and various amounts of MT standard were electrophoresed (constant current, 20 mA per slab) for 9 hours. Gels were stained with Commassie Blue (R-250, 0.25%) for 12 hours and destained. Gels were scanned by densitometer and peaks heights were determined. Significantly linear standard curves (..mu..g MT vs. peak height) were established for both MTI and MTII. (Cd, Zn)-MTI migrated slower than Zn-MTI while mobilities for both (Cd, Zn)- and Zn-MTII were the same. The accumulation of MTI was consistently less than MTII in liver from both Zn- and Cd-injected rats. Their results suggest that electrophoretic analysis is an excellent system not only for quantitation but also for characterization of MT in rat tissue.

  13. Development of a microfluidics-based gel protein recovery system.

    PubMed

    Razunguzwa, Trust Tariro; Biddle, April; Anderson, Heather; Zhan, Dongliang; Powell, Matthew

    2009-12-01

    A PMMA microfluidic chip, in which fluid is manipulated to transport protein from a PAGE gel piece to a collection reservoir via a microfluidic channel, has been developed. The protein sample is mobilized out of the gel (loaded in a chip access hole) into a low EOF-CE microfluidic channel under the influence of an electric field. Simultaneously, hydrostatic pressure from the filled buffer reservoirs is used to direct the protein sample to a third reservoir, through a field-free channel connected to the electrophoresis channel. Using this novel process of protein transport from a gel sample, proteins from Coomassie-stained gels have been transferred into solution in 15-30 min, with good sample recovery, using a run buffer containing an anionic acid-labile surfactant. A variety of small- and medium-sized proteins were successfully recovered and detected using both electrospray and MALDI MS over gel loads of 0.1-10 microg. This technological tool is very important for extracting quality intact protein samples from polyacrylamide gels, from which accurate protein molecular weights and protein sequences can be obtained using intact molecules. PMID:19960466

  14. Programs for Native People by Native People

    ERIC Educational Resources Information Center

    Kirkness, Verna J.

    1976-01-01

    Education programs for native people have become the target of serious criticism. Describes the perspective of Canadian Indians in terms of educational philosophy and educational objectives. (Author/RK)

  15. Silver staining of proteins in 2DE gels.

    PubMed

    Lelong, Cécile; Chevallet, Mireille; Luche, Sylvie; Rabilloud, Thierry

    2009-01-01

    Silver staining detects proteins after electrophoretic separation on polyacrylamide gels. Its main positive features are its excellent sensitivity (in the low nanogram range) and the use of very simple and cheap equipment and chemicals. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation, and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks. PMID:19381593

  16. Polyacrylamide hydrogel injection for breast augmentation: Another injectable failure

    PubMed Central

    Wang, Zhenxiang; Li, Shirong; Wang, Lingli; Zhang, Shu; Jiang, Yan; Chen, Jinping; Luo, Donglin

    2012-01-01

    Summary Background Increasing complications of polyacrylamide hydrogel (PAAG) augmentation mammoplasty, such as chronic persistent infection, have recently caught the attention of both the medical field and the general public. Material/Methods A total of 96 patients with severe chronic infection following PAAG augmentation mammoplasty were treated in the present study including 63 cases with infection confined to the breast and 33 with systemic infection. Endoscopy and surgery were performed to completely remove the materials and clear the infected tissues followed by drug-irrigation and vacuum-assisted closure for several days. Results In patients with severe infection there were large amounts of PAAG, fibers and infiltration of numerous neutrophils and macrophages. The infection-inducing materials were extensively dispersed in the mammary and subcutaneous tissues, pectoral fascia and intermuscular space. In addition, there was scattered distribution of PAAG materials in the armpit, chest wall and abdominal wall, which were mixed with necrotic tissues and surrounded by lymphocytes, giant cells, macrophages and other inflammatory cells, forming chronic granulomatous and fibrous lesions. Infection was controlled following surgical intervention. No residual infectious foci or recurrent infections were noted among these patients. Although the severe infection did not result in mastectomy, patients had breast atrophy and various degrees of deformation. Conclusions Chronic infection following PAAG augmentation mammaplasty usually causes systemic infection and other devastating adverse reactions. This study confirms PAAG augmentation mammaplasty is another failed attempt. More attention should be paid to the injection of large doses of liquid filler. PMID:22648256

  17. Effect of cationic polyacrylamides on the interactions between cellulose fibers.

    PubMed

    Mosse, Wade K J; Boger, David V; Simon, George P; Garnier, Gil

    2012-02-21

    The interaction between cellulose fibers in the presence of cationic polyacrylamide (CPAM) was analyzed by rheology as a function of polyelectrolyte concentration, charge density, and molecular weight. CPAM was found to strongly influence the yield stress of cellulose suspensions; low doses of CPAM increased the yield stress, but at higher concentrations the yield stress declined. The charge density of the CPAM was the most significant factor in how yield stress responded to CPAM concentration; this effect was able to be normalized to a master curve by considering only the charged fraction of the polymer. The molecular weight of CPAM samples had some effect at high concentrations, but for lower CPAM doses the yield stress was independent of molecular weight over the range studied. The data suggest that CPAM modifies the interaction between cellulose surfaces via several mechanisms, with electrostatic interactions in the form of charge neutralization and charged patch formation dominating; polymer bridging and steric repulsion also influence the overall balance of forces between interacting cellulose fibers. PMID:22316050

  18. Polyacrylamide medium for the electrophoretic separation of biomolecules

    DOEpatents

    Madabhushi, Ramakrishna S.; Gammon, Stuart A.

    2003-11-11

    A polyacryalmide medium for the electrophoretic separation of biomolecules. The polyacryalmide medium comprises high molecular weight polyacrylamides (PAAm) having a viscosity average molecular weight (M.sub.v) of about 675-725 kDa were synthesized by conventional red-ox polymerization technique. Using this separation medium, capillary electrophoresis of BigDye DNA sequencing standard was performed. A single base resolution of .about.725 bases was achieved in .about.60 minute in a non-covalently coated capillary of 50 .mu.m i.d., 40 cm effective length, and a filed of 160 V/cm at 40.degree. C. The resolution achieved with this formulation to separate DNA under identical conditions is much superior (725 bases vs. 625 bases) and faster (60 min. vs. 75 min.) to the commercially available PAAm, such as supplied by Amersham. The formulation method employed here to synthesize PAAm is straight-forward, simple and does not require cumbersome methods such as emulsion polymerizaiton in order to achieve very high molecular weights. Also, the formulation here does not require separation of PAAm from the reaction mixture prior to reconstituting the polymer to a final concentration. Furthermore, the formulation here is prepared from a single average mol. wt. PAAm as opposed to the mixture of two different average mo. wt. PAAm previously required to achieve high resolution.

  19. Content in Native Literature Programs.

    ERIC Educational Resources Information Center

    Grant, Agnes

    Including Native literature in school curricula is an important way of enhancing the Native student's self-concept and providing accurate Native cultural knowledge to Native and non-Native students alike. Nevertheless, Canadian school literature programs generally contain neither contemporary nor traditional Native literature. Some programs…

  20. DEVELOPMENT OF POLYMER GEL SYSTEMS TO IMPROVE VOLUMETRIC SWEEP AND REDUCE PRODUCING WATER/OIL RATIOS

    SciTech Connect

    G. Paul Willhite; Stan McCool; Don W. Green; Min Cheng; Rajeev Jain; Tuan Nguyen

    2003-11-01

    Gelled polymer treatments are applied to oil reservoirs to increase oil production and to reduce water production by altering the fluid movement within the reservoir. This report describes the results of the first year of a three-year research program that is aimed at the understanding of the chemistry of gelation and the fundamental mechanisms that alter the flows of oil and water in reservoir rocks after a gel treatment. Work has focused on a widely-applied system in field applications, the partially hydrolyzed polyacrylamide-chromium acetate gel. Gelation occurs by network formation through the crosslinking of polyacrylamide molecules as a result of reaction with chromium acetate. The initial reaction between chromium acetate and one polymer is referred to as the uptake reaction. The uptake reaction was studied as functions of chromium and polymer concentrations and pH values. Experimental data were regressed to determine a rate equation that describes the uptake reaction of chromium by polyacrylamide. Pre-gel aggregates form and grow as the reactions between chromium acetate and polyacrylamide proceed. A statistical model that describes the growth of pre-gel aggregates was developed using the theory of branching processes. The model gives molecular weight averages that are expressed as functions of the conversion of the reactive sites on chromium acetate or on the polymer molecule. Results of the application of the model correlate well with experimental data of viscosity and weight-average molecular weight and gives insights into the gelation process. A third study addresses the flow of water and oil in rock material after a gel treatment. Previous works have shown that gel treatments usually reduce the permeability to water to a greater extent than the permeability to oil is reduced. This phenomenon is referred to as disproportionate permeability reduction (DPR). Flow experiments were conducted to determine the effect of polymer and chromium concentrations on DPR. All gels studied reduced the permeability to water by a greater factor than the factor by which the oil permeability was reduced. Greater DPR was observed as the concentrations of polymer and chromium were increased. Increased pressure gradients during oil flow decreased the oil permeability and the water permeability that was measured afterward. Lower pressure gradients that were applied subsequently moderately affected water permeabilities but did not affect oil permeabilities. A conceptual model of the mechanisms responsible for DPR is presented. Primary features of the model are (1) the development of flow channels through the gel by dehydration of the gel and by re-connection of pre-treatment, residual oil volume and (2) high flow resistance in the channels during water flow is caused by significant saturations of oil remaining in the channels.

  1. Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.

    PubMed

    Wrigley, C W; Margolis, J

    1992-01-01

    Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow. PMID:1599958

  2. 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.

  3. Elastic properties of swollen polyelectrolyte gels in aqueous salt solutions

    NASA Astrophysics Data System (ADS)

    Sasaki, Shigeo

    2006-03-01

    The elastic relaxation responding to a uniaxially stretched poly(acrylic acid) rodlike gel in the aqueous NaCl solution was investigated. The relaxation elucidated the shear (μ) and bulk (K) moduli and the frictional coefficients (ς) of the fully ionized gel at pH above 9 as functions of the degree of swelling, which was controlled by the NaCl concentration (CS) of the solution. Two gels, cross-linked chains of which consist of 500 (GelA500) and 50 (GelA50) monomeric units, were examined to investigate the effect of the chain length on the elastic behavior. The moduli of GelA500 increased with swelling at CS below 100 mM and decreased at CS above it. The μ values of both gels can be characterized by the power function of gel diameter, d as μ ∝dβ. The β values being -1 at CS above 100 mM transitionally changed to 1.2 at CS about 100 mM. That is, the dimensionality of space for the chains to distribute, ndim {=(β+5)/(β+2) according to the conventional theory [Sasaki et al., J. Chem. Phys. 102, 5694 (1995)]}, changed from 4 (ndim of ideal chain) to 1.9 at CS=100mM. This indicates that the electrostatic repulsion between gel chains reduces the ndim at CS below 100 mM but not at CS above it. It was found that the K values of GelA500 were well described by the conventional theory but those of GelA50 not. The ςzr/ςrr (ςzr is an off-diagonal element of the friction coefficient tensor and ςrr is a diagonal element of the friction coefficient tensor) was found to be 2.7×10-2, which was the same as the previously reported value for the swollen poly(acrylamide) gel [Sasaki, J. Chem. Phys. 120, 5789 (2004)]. The ςrr of unit volume of the GelA500 is well explained by the free draining model while the ςrr of GelA50 is less than that of the free draining model, suggesting the slip between water and ionized chain.

  4. Waste-Activated Sludge Fermentation for Polyacrylamide Biodegradation Improved by Anaerobic Hydrolysis and Key Microorganisms Involved in Biological Polyacrylamide Removal

    PubMed Central

    Dai, Xiaohu; Luo, Fan; Zhang, Dong; Dai, Lingling; Chen, Yinguang; Dong, Bin

    2015-01-01

    During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM. PMID:26144551

  5. Waste-Activated Sludge Fermentation for Polyacrylamide Biodegradation Improved by Anaerobic Hydrolysis and Key Microorganisms Involved in Biological Polyacrylamide Removal.

    PubMed

    Dai, Xiaohu; Luo, Fan; Zhang, Dong; Dai, Lingling; Chen, Yinguang; Dong, Bin

    2015-01-01

    During the anaerobic digestion of dewatered sludge, polyacrylamide (PAM), a chemical conditioner, can usually be consumed as a carbon and nitrogen source along with other organic matter (e.g., proteins and carbohydrates in the sludge). However, a significant accumulation of acrylamide monomers (AMs) was observed during the PAM biodegradation process. To improve the anaerobic hydrolysis of PAM, especially the amide hydrolysis process, and to avoid the generation of the intermediate product AM, a new strategy is reported herein that uses an initial pH of 9, 200 mg COD/L of PAM and a fermentation time of 17 d. First, response surface methodology (RSM) was applied to optimize PAM removal in the anaerobic digestion of the sludge. The biological hydrolysis of PAM reached 86.64% under the optimal conditions obtained from the RSM. Then, the mechanisms for the optimized parameters that significantly improved the biological hydrolysis of PAM were investigated by the synergistic effect of the main organic compounds in the sludge, the floc size distribution, and the enzymatic activities. Finally, semi-continuous-flow experiments for a microbial community study were investigated based on the determination of key microorganisms involved in the biological hydrolysis of PAM. PMID:26144551

  6. Investigation of the properties of polyacrylamide-polyaniline composite and its application as a battery electrode

    SciTech Connect

    Bhat, N.V.; Joshi, N.V. . Dept. of Chemical Technology)

    1993-11-20

    The composite films of polyacrylamide and polyaniline were prepared by polymerizing aniline using ammonium persulfate as an initiator in an aqueous solution containing poly-acrylamide. A film was then cast from this solution. The structural, dynamic mechanical, electrical, and thermal properties of these films have been studied. The infrared spectrum shows the presence of polyacrylamide as well as polyaniline in the composite film. The thermal analysis shows that the composite degrades slower than does the polyacrylamide alone. The dynamic mechanical analysis indicates that there is an increase in the glass transition temperature after the composite formation. The electrical conductivity has been found to increase by more than eight orders of magnitude. These composite films have also been suitably used as electrodes in secondary batteries.

  7. 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.

  8. 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.

  9. Check dam and polyacrylamide performance under simulated stormwater runoff.

    PubMed

    Kang, Jihoon; McCaleb, Melanie M; McLaughlin, Richard A

    2013-11-15

    High levels of turbidity and fine suspended sediments are often found in stormwater discharges from construction sites even when best management practices (BMPs) for sediment control are in place. This study evaluated turbidity reduction by three check dam types: 1) rock check dam representing a standard BMP, 2) excelsior wattle representing a fiber check dam (FCD), and 3) rock check dam wrapped with excelsior erosion control blanket (rock + excelsior ECB) representing an alternative FCD. Three check dams (all same type) were installed in a lined, 24-m ditch on a 5-7% slope and three consecutive simulated stormwater flows were run in the ditch. Additional tests were performed by adding granular polyacrylamide (PAM) on the check dams in the same manner using two sediment sources differing in clay content. Without PAM treatment, significantly higher effluent turbidity (>900 nephelometric turbidity units (NTU)) exited the ditch with rock check dams than with excelsior wattles or rock + excelsior ECBs (<440 NTU). The extent of sediment deposition between the check dam types was in the order of excelsior wattle > rock + excelsior ECB > rock check dam, indicating better water pooling behind the wattle. The PAM treatment reduced turbidity substantially (>75% relative to no PAM treatment) for all check dam types and it was very effective in excelsior wattles (<57 NTU) and rock + excelsior ECBs (<90 NTU) even during the third storm event. This study demonstrates that the passive treatment of runoff with PAM on FCDs (or rock + excelsior ECB) in construction site ditches can be very effective for sediment retention and turbidity reduction. PMID:24036092

  10. Implicit Attitudes towards Native and Non-Native Speaker Teachers

    ERIC Educational Resources Information Center

    Todd, R. Watson; Pojanapunya, Punjaporn

    2009-01-01

    The academic literature and educational principle suggest that native and non-native English speaking teachers should be treated equally, yet in many countries there is a broad social and commercial preference for native speaker teachers which may also involve racial issues. Attitudes towards native and non-native English speaking teachers have

  11. Light scattering from a binary-liquid entanglement gel

    NASA Astrophysics Data System (ADS)

    Xia, K.-Q.; Maher, J. V.

    1987-09-01

    Light-scattering experiments have been carried out on an entanglement gel with a binary-liquid mixture as solvent. The onset temperature for critical opalescence has a composition dependence which is similar to the coexistence curve of the free-liquid mixture. This system resembles previously reported work on the cross-linked gel polyacrylamide in two ways: (1) As temperature is lowered toward the critical temperature of the free-liquid mixture, the binary-fluid gel exhibits a strong and increasing light scattering over a broad temperature region of several kelvins, and (2) no appreciable temporal fluctuations are observed throughout this temperature region. Two added features are observed in the present, entanglement-gel measurements: (a) Gel samples with solvent composition both near and off the critical composition of the free-liquid mixture exhibit similar light-scattering behavior, and (b) a Lorentzian-squared fit to the light-scattering angular distributions yields a characteristic wave number which does not change with temperature and an amplitude which shows a very strong dependence on the temperature.

  12. Protein gel staining methods: an introduction and overview.

    PubMed

    Steinberg, Thomas H

    2009-01-01

    Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on suitable protein gel staining and detection methods. A total protein profile can be determined with the colorimetric methods embodied in Coomassie Blue and silver staining methods, or increasingly, with fluorescent stains. Protein quantitation can be done following staining, with fluorescence- and instrumentation-based methods offering the greatest sensitivity and linear dynamic range. Protein posttranslational modifications such as phosphorylation and glycosylation can be reliably determined with several fluorescence-based protocols. Staining and detection with two or more different stains can be done in series to establish relative profiles of modified versus total protein or to assess purity at two levels of quantitative sensitivity. The choice of staining method and protocol depends on the required rigor of detection and quantitation combined with available instrumentation and documentation capabilities. Other considerations for staining methods include intended downstream analytical procedures such as mass spectrometry or peptide sequencing, which preclude some methods. Nonfixative staining methods allow western blotting after gel staining. Laboratory custom and budget or intellectual curiosity may be the ultimate determinate of the chosen gel staining protocol. PMID:19892191

  13. Gold Colloid Dynamics in Polymer Solutions and Gels

    NASA Astrophysics Data System (ADS)

    Reina, J. C.; Hope, D.

    1998-03-01

    The dependence of the dynamics of gold colloid nanospheres in polyacrylamide solutions and gels as a function of crosslink content and scattering angle is studied using dynamic light scattering methods. Auto-correlation functions spanning up to nine sampling time scales were measured from samples ranging through the gel threshold. Cumulant and Inverse Laplace Transform fitting methods indicate a complex dynamical behavior ranging from the purely translational Brownian diffusion of the nanospheres in the medium, through a regime of wide distribution of relaxation rates. Well beyond the gelation threshold, where a large fraction of the gold particles are trapped, we observe a coupling between the movement of the particles and the collective diffusion mode of the polymer network itself. The crossover from diffusional to relaxational behavior depends on the length scale of the probe particles.

  14. Polyacrylamide + Al2(SO4)3 and polyacrylamide + CaO remove coliform bacteria and nutrients from swine wastewater.

    PubMed

    Entry, James A; Phillips, Ian; Stratton, Helen; Sojka, R E

    2003-01-01

    Animal wastes are a major contributor of nutrients and enteric microorganisms to surface water and ground water. Polyacrylamide (PAM) mixtures are an effective flocculent, and we hypothesized that they would reduce transport of microorganisms in flowing water. After waste water running at 60.0 1 min(-1) flowed over PAM + Al2(SO4)3, or PAM + CaO in furrows, total coliform bacteria (TC) and fecal coliform bacteria (FC) were reduced by 30-50% at 1 and 50 m downstream of the treatments compared to the control. In a column study, PAM + Al2(SO4)3, and PAM + CaO applied to sandy, sandy loam, loam, and clay soils reduced NH4+ and ortho-P concentrations in leachate compared to the source waste water and the control. PAM + Al2(SO4)3 and PAM + CaO applied to sandy, sandy loam and loam soils reduced both total and ortho-P, concentrations in leachate compared to he source wastewater and control treatment. In a field study, PAM + Al2(SO4)3, or PAM + CaO treatments did not consistently reduce NH4+, NO3-, ortho-P, and total P concentrations in wastewater flowing over any soil compared to inflow wastewater or the control treatment. With proper application PAM + Al2(SO4)3 and PAM + CaO may be able to reduce the numbers of enteric bacteria in slowly flowing wastewater running off animal confinement areas, reducing the amount of pollutants entering surface water and groundwater. PMID:12685771

  15. Tionantati: Native Tobacco People.

    ERIC Educational Resources Information Center

    Shorty, Lawrence

    1997-01-01

    Recounts the author's, and his family's, relationship with tobacco. Identifies tobacco's role in traditional Native American ceremonies as encouraging communication and self-reflection. Describes Tionantati: Native Tobacco People, an intervention program that returns tobacco to its traditional use and eschews recreational use of commercial

  16. AIDS and Native Youth.

    ERIC Educational Resources Information Center

    Luna, G. Cajetan

    Native Americans throughout North America suffer from a greater prevalence of health problems than the population as a whole. One might believe that the problem of AIDS is insignificant for Native youth, but such a belief is inaccurate and shortsighted. As of March 1989, the Centers for Disease Control reported 1,792 cases of childhood and…

  17. Listen to the Natives

    ERIC Educational Resources Information Center

    Prensky, Marc

    2006-01-01

    "Digital natives" refer to today's students because they are native speakers of technology, fluent in the digital language of computers, video games, and the Internet. Those who were not born into the digital world are referred to as digital immigrants. Educators, considered digital immigrants, have slid into the 21st century--and into the digital…

  18. Native American Entrepreneurship. Digest.

    ERIC Educational Resources Information Center

    Seymour, Nicole

    Although Native Americans have owned and started the fewest small businesses of all U.S. minority groups, entrepreneurship is considered to be an efficient tool for alleviating their economic problems. Barriers to Native American entrepreneurship include poverty, scarce start-up capital, poor access to business education and technical assistance,…

  19. Native American Preparatory School.

    ERIC Educational Resources Information Center

    Native American Preparatory School, Rowe, NM.

    This booklet provides information on the Native American Preparatory School, a residential secondary school in Rowe, New Mexico, for high-achieving Native American students. The school sponsors two programs: a 5-week rigorously academic summer school for junior high school students and, beginning in fall 1995, a 4-year college preparatory program.…

  20. Traditional Native Poetry.

    ERIC Educational Resources Information Center

    Grant, Agnes

    1985-01-01

    While Native myths and legends were educational tools to transmit tribal beliefs and history, traditional American Indian poetry served a ritualistic function in everyday life. Few traditional Native songs, which all poems were, survive; only Mayan and Aztec poems were written, and most of these were burned by a Spanish bishop. In addition, many…

  1. Fluid diversion and sweep improvement with chemical gels in oil recovery processes. Final report

    SciTech Connect

    Seright, R.S.; Martin, F.D.

    1992-09-01

    The objectives of this project were to identify the mechanisms by which gel treatments divert fluids in reservoirs and to establish where and how gel treatments are best applied. Several different types of gelants were examined, including polymer-based gelants, a monomer-based gelant, and a colloidal-silica gelant. This research was directed at gel applications in water injection wells, in production wells, and in high-pressure gas floods. The work examined how the flow properties of gels and gelling agents are influenced by permeability, lithology, and wettability. Other goals included determining the proper placement of gelants, the stability of in-place gels, and the types of gels required for the various oil recovery processes and for different scales of reservoir heterogeneity. During this three-year project, a number of theoretical analyses were performed to determine where gel treatments are expected to work best and where they are not expected to be effective. The most important, predictions from these analyses are presented. Undoubtedly, some of these predictions will be controversial. However, they do provide a starting point in establishing guidelines for the selection of field candidates for gel treatments. A logical next step is to seek field data that either confirm or contradict these predictions. The experimental work focused on four types of gels: (1) resorcinol-formaldehyde, (2) colloidal silica, (3) Cr{sup 3+}(chloride)-xanthan, and (4) Cr{sup 3+}(acetate)-polyacrylamide. All experiments were performed at 41{degrees}C.

  2. Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci.

    PubMed

    Chen, Xiao-he; O'Dell, Sandra D; Day, Ian N M

    2002-05-01

    After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment. PMID:12019781

  3. Federal Politics and Native Concerns. Native Viewpoints.

    ERIC Educational Resources Information Center

    Steinhauer, Noella

    1997-01-01

    Addresses some of the frustration felt by Canada's indigenous Indian population regarding the lack of attention afforded them in recent federal elections. Discusses Indian reaction to the Canadian Government's White Paper on Aboriginal peoples. Canadian natives felt this document actually represented a step backward in their struggle for equality.…

  4. Native SAD is maturing

    PubMed Central

    Rose, John P.; Wang, Bi-Cheng; Weiss, Manfred S.

    2015-01-01

    Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow ‘highly accurate data’ to be routinely collected. Today, native SAD sits on the verge of becoming a ‘first-choice’ method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development. PMID:26175902

  5. Native American Studies and Curriculum.

    ERIC Educational Resources Information Center

    Dorris, Michael A.

    The Native Studies curriculum has an important role to play in the development of meaningful community control for Native American education. Native Studies may legitimately take many forms and employ many methods; the essential element is that educators adopt a clear and consistent attitude toward Native Studies based on respect for Native people…

  6. Solids and nutrient removal from flushed swine manure using polyacrylamides

    SciTech Connect

    Vanotti, M.B.; Hunt, P.G.

    1999-12-01

    Most of the organic nutrients and reduced carbon (C) materials in liquid swine manure are contained in fine suspended particles that are not separated by available mechanical separators. Treatment with polyacrylamide (PAM) polymers prior to mechanical removal or gravity settling has the potential for enhancing solids-liquid separation, thus concentrating nitrogen (N), phosphorus (P), and organic C. In this work, the authors determined PAM charge and density characteristics most desirable for swine wastewater applications and established the optimum chemical requirement. Treatments were applied to flushed manure from two swine operations in North Carolina. Cationic PAMs significantly increased solids separation while performance of neutral and anionic types was not different from a control. Cationic PAMs with moderate-charge density (20%) were more effective than polymers with higher charge density. Flocs were large and effectively retained with a 1-mm screen. Optimum PAM rate varied with the amount of total suspended solids (TSS) in the liquid manure; 26 and 79 mg PAM/L for samples containing 1.5 and 4.1 g TSS/L, respectively. Corresponding TSS removal efficiencies were 90 to 94%. In contrast, screening without PAM treatment captured only 5 to 14% of the suspended solids. Polymer usage rate was consistent and averaged 2.0{degree} based on weight of dry solids produced. Volatile suspended solids (VSS) were highly correlated with TSS and comprised 79.5% of TSS. Chemical oxygen demand (COD) and organic nutrient concentrations in the effluent were also significantly decreased by PAM treatment. The decrease of COD concentration, an important consideration for odor control, was linearly related with removal of suspended solids, at a rate of 2.0 g COD/g TSS and 2.6 g COD/g VSS. Removal efficiency of organic N and P followed approximately a 1:1 relationship with removal efficiency of TSS. Chemical cost to capture 90% of the suspended solids was estimated to be $0.026 per hog per day ($2.79 per finished hog). Results obtained indicate that PAM treatment is very effective for removal of manure solids, COD, and organic nutrients from flushed swine effluents. The technology provides an attractive alternative to existing liquid manure handling methods for conserving nutrients and avoiding excessive nutrient application in areas where swine production is concentrated.

  7. Sol-Gel Glasses

    NASA Technical Reports Server (NTRS)

    Mukherjee, S. P.

    1985-01-01

    Multicomponent homogeneous, ultrapure noncrystalline gels/gel derived glasses are promising batch materials for the containerless glass melting experiments in microgravity. Hence, ultrapure, homogeneous gel precursors could be used to: (1) investigate the effect of the container induced nucleation on the glass forming ability of marginally glass forming compositions; and (2) investigate the influence of gravity on the phase separation and coarsening behavior of gel derived glasses in the liquid-liquid immiscibility zone of the nonsilicate systems having a high density phase. The structure and crystallization behavior of gels in the SiO2-GeO2 as a function of gel chemistry and thermal treatment were investigated. As are the chemical principles involved in the distribution of a second network former in silica gel matrix being investigated. The procedures for synthesizing noncrystalline gels/gel-monoliths in the SiO2-GeO2, GeO2-PbO systems were developed. Preliminary investigations on the levitation and thermal treatment of germania silicate gel-monoliths in the Pressure Facility Acoustic Levitator were done.

  8. Improvement in the accuracy of polymer gel dosimeters using scintillating fibers

    NASA Astrophysics Data System (ADS)

    Tremblay, Nicolas M.; Hubert-Tremblay, Vincent; Bujold, Rachel; Beaulieu, Luc; Lepage, Martin

    2010-11-01

    We propose a novel method for the absolute calibration of polyacrylamide gel (PAG) dosimeters with one or more reference scintillating fiber dosimeters inserted inside the gel. Four calibrated scintillating fibers were inserted into a cylindrical glass container filled with a PAG dosimeter irradiated with a wedge filtered 6 MV photon beam. Calibration curves using small glass vials containing the same gel as the cylindrical containers were used to obtain a first calibration curve. This calibration curve was then adjusted with the dose measured with one of the scintillating fibers in a low gradient part of the field using different approaches. Among these, it was found that a translation of the gel calibration curve yielded the highest accuracy with PAG dosimeters.

  9. Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

    PubMed Central

    2010-01-01

    The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions. PMID:21406065

  10. Swelling and shrinking of a polyelectrolyte gel induced by a salt solution

    NASA Astrophysics Data System (ADS)

    Rydzewski, R.

    1990-05-01

    The stability of a polyelectrolyte gel in solution results from a delicate balance between several competing thermodynamic forces, viz. (i) osmotic pressure of free ions in the gel, (ii) molecular interaction of solvent and polymer molecules, (iii) network elasticity, (iv) Debye-Htickel interaction of ions. That balance may be upset by a decrease of temperature and by the addition of salt to the solvent. This results in a decrease of osmotic pressure and collapse of the gel to a small fraction of the initial volume. The effect can be reversed by increasing temperature and by removing salt from the solution. This paper presents an attempt to describe swelling and shrinking quantitatively and to understand the nature of the opposing forces. The volume of a particular polyacrylamide gel in a water acetone solution is represented as a function of the salt content and of temperature.

  11. Development of Polymer Gel Systems to Improve Volumetric Sweep and Reduce Producing Water/Oil Ratios

    SciTech Connect

    G. Paul Willhite; Stan McCool; Don W. Green; Min Cheng; Feiyan Chen

    2005-04-03

    Gelled polymer treatments are applied to oil reservoirs to increase oil production and to reduce water production by altering the fluid movement within the reservoir. This report describes the results of the third year of a 42 month research program that is aimed at an understanding of gelation chemistry and the fundamental mechanisms that alter the flows of oil and water in reservoir rocks after a gel treatment. Work focused on a widely applied system in the field, the partially hydrolyzed polyacrylamide-chromium acetate gel. Gelation occurs by network formation through the crosslinking of polyacrylamide molecules as a result of reaction with chromium acetate. Pre-gel aggregates form and grow as reactions between chromium acetate and polyacrylamide proceed. A mathematical model that describes uptake and crosslinking reactions as a function of time was derived. The model was probability based and provides molecular-weight averages and molecular-weight distributions of the pre-gel aggregates as a function of time and initial system conditions. A liquid chromatography apparatus to experimentally measure the size and molecular weight distributions of polymer samples was developed. The method worked well for polymer samples without the chromium crosslinker. Sample retention observed during measurements of gelant samples during the gelation process compromised the results. Other methods will be tested to measure size distributions of the pre-gel aggregates. Dissolution of carbonate minerals during the injection of gelants causes the pH of the gelant to increase. Chromium precipitates from solution at the higher pH values robbing the gelant of crosslinker. Experimental data on the transport of chromium acetate solutions through dolomite cores were obtained. A mathematical model that describes the transport of brine and chromium acetate solutions through rocks containing carbonate minerals was used to simulate the experimental results.

  12. Pulse Field Gel Electrophoresis.

    PubMed

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

    2016-01-01

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

  13. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2003-09-01

    This report describes work performed during the second year of the project, ''Conformance Improvement Using Gels.'' The project has two objectives. The first objective is to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective is to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil. Pore-level images from X-ray computed microtomography were re-examined for Berea sandstone and porous polyethylene. This analysis suggests that oil penetration through gel-filled pores occurs by a gel-dehydration mechanism, rather than a gel-ripping mechanism. This finding helps to explain why aqueous gels can reduce permeability to water more than to oil. We analyzed a Cr(III)-acetate-HPAM gel treatment in a production well in the Arbuckle formation. The availability of accurate pressure data before, during, and after the treatment was critical for the analysis. After the gel treatment, water productivity was fairly constant at about 20% of the pre-treatment value. However, oil productivity was stimulated by a factor of 18 immediately after the treatment. During the six months after the treatment, oil productivity gradually decreased to approach the pre-treatment value. To explain this behavior, we proposed that the fracture area open to oil flow was increased substantially by the gel treatment, followed by a gradual closing of the fractures during subsequent production. For a conventional Cr(III)-acetate-HPAM gel, the delay between gelant preparation and injection into a fracture impacts the placement, leakoff, and permeability reduction behavior. Formulations placed as partially formed gels showed relatively low pressure gradients during placement, and yet substantially reduced the flow capacity of fractures (with widths from 1 to 4 mm) during brine and oil flow after placement. Regardless of gel age before placement, very little gel washed out from the fractures during brine or oil flow. However, increased brine or oil flow rate and cyclic injection of oil and water significantly decreased the level of permeability reduction. A particular need exists for gels that can plug large apertures (e.g., wide fractures and vugs). Improved mechanical strength and stability were demonstrated (in 1- to 4-mm-wide fractures) for a gel that contained a combination of high- and low-molecular weight polymers. This gel reduced the flow capacity of 2- and 4-mm-wide fractures by 260,000. In a 1-mm-wide fracture, it withstood 26 psi/ft without allowing any brine flow through the fracture. Cr(III)-acetate-HPAM gels exhibited disproportionate permeability reduction in fractures. The effect was most pronounced when the gel was placed as gelant or partially formed gels. The effect occurred to a modest extent with concentrated gels and with gels that were ''fully formed'' when placed. The effect was not evident in tubes. We explored swelling polymers for plugging fractures. Polymer suspensions were quickly prepared and injected. In concept, the partially dissolved polymer would lodge and swell to plug the fracture. For three types of swelling polymers, behavior was promising. However, additional development is needed before their performance will be superior to that of conventional gels.

  14. Synthesis of polyacrylamide-montmorillonite clay nanocomposite using non-conventional electrochemical technique.

    PubMed

    Srivastava, Monika; Prakash, Rajiv

    2012-01-01

    Synthesis of polymer-clay nanocomposite by in-situ incorporation of polyacrylamide in organically modified montmorillonite (MMT) clay layers is being reported using non-conventional electrochemical technique "plasma electrolysis." A luminous sheath of plasma is sustained between an electrode (anode) and the surface of surrounding liquid electrolyte at sufficiently high voltage, for synthesis of polymer or nanocomposite. Using this technique, radical generation capability is explored as a new tool for radical polymerization and in-situ composite formation of polyacrylamide. Polyacrylamide-MMT clay nanocomposite is synthesized by taking acrylamide and MMT clay in K2SO4 electrolyte solution at the anodic potential of 660 V. Polyacrylamide and polyacrylamide-MMT clay nanocomposites are characterized for their structural and thermal properties. Intercalation in MMT clay layers of homogeneous nanocomposite is supported by X-ray diffraction, FTIR, DSC, TGA and SEM/TEM techniques. This novel method produces homogeneous interactive composite with high yield, and shows potential to replace chemical initiators based harsh synthetic processes used for conventional polymer-nanocomposites formation. PMID:22524007

  15. Gel properties of sutchi catfish (Pangasius hypophthalmus) surimi as affected by selected washing process and number of washing cycles.

    PubMed

    Priyadarshini, Bhargavi; Majumdar, Ranendra K; Parhi, Janmejay; Maurya, Pradip K; Roy, Deepayan; Saha, Apurba

    2016-04-01

    The gel properties of sutchi catfish surimi prepared by conventional washing and alkaline-saline washing method were studied for four washing cycles. Decrease (p < 0.05) in myoglobin content was found in alkaline washing process compared to conventional washing at each washing cycle. The highest hardness, breaking force and deformation was observed in gels prepared from alkaline-saline washing method. Whiteness in conventional washed surimi gels increased non-significantly (p < 0.05) compared to alkaline-saline-washed surimi gels. Protein bands on Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the protein extractability was more in alkaline-saline washing and disappearance of bands in conventional washing method was observed between 116 and 45 kDa indicating less yield of protein. PMID:26199223

  16. High-Frequency Alternating-Crossed-Field Gel Electrophoresis WithNeutral or Slightly Charged Interpenetrating Networks to Improve DNASeparation

    SciTech Connect

    Boyd, B.; Prausnitz, J.; Blanch, H.

    1998-07-01

    Toward improving DNA separations, this work reports theeffects of high-frequency square-wave AC fields superimposedperpendicular to the direct current (DC) separation field on DNAmigration in both polyacrylamide-based interpenetrating networks (IPNs)and in agarose networks. Compared to standard polyacrylamide gels, IPNsallow the separation of larger DNA (9000 bp vs. 5000 bp at 5 V/cm). Innovel polyacrylamide-based IPNs, an alternating current (AC) field of 5Hz increased the maximum DNA size separable. This effect was extended tolarger DNA sizes with increasing electric-field strength up to andapparently beyond the power supply-limited maximum electric-fieldstrength of 48 V/cm. The orthogonal AC field also increased mobility.These two results combine to yield a reduction in separation time of upto a factor of 20 in novel polyacrylamide-based IPNs. When negativelycharged acrylic-acid groups were incorporated into the IPNs, the use ofthe AC field changed the DNA-network interaction, which altered the sizedependence of DNA mobility. In agarose gels, an AC field of 50 Hzincreased the size range separable; however, there was no increase in DNAmobility. There was no change in size dependence of mobility in an ACfield when the number of charged groups in the agarose network wasincreased. Based on results in the literature, possible mechanisms wereexamined for the effects of the AC field on DNA separation.

  17. Native American Tribal Websites.

    ERIC Educational Resources Information Center

    Miller, Eric L.

    1999-01-01

    Lists Web sites maintained by 38 different Native American nations that deal with topics ranging from tribal history, news, arts and crafts, tourism, entertainment, and commerce. Represented nations include Apache, Blackfeet, Creek, Iroquois, Mohegan, and Sioux. (CMK)

  18. Native American Identity

    ERIC Educational Resources Information Center

    Horse, Perry G.

    2005-01-01

    Many issues and elements--including ethnic nomenclature, racial attitudes, and the legal and political status of American Indian nations and Indian people--influence Native American identity. (Contains 3 notes.)

  19. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2004-09-30

    This report describes work performed during the third and final year of the project, ''Conformance Improvement Using Gels.'' Corefloods revealed throughput dependencies of permeability reduction by polymers and gels that were much more prolonged during oil flow than water flow. This behavior was explained using simple mobility ratio arguments. A model was developed that quantitatively fits the results and predicts ''clean up'' times for oil productivity when production wells are returned to service after application of a polymer or gel treatment. X-ray computed microtomography studies of gels in strongly water-wet Berea sandstone and strongly oil-wet porous polyethylene suggested that oil penetration through gel-filled pores occurs by a gel-dehydration mechanism, rather than gel-ripping or gel-displacement mechanisms. In contrast, analysis of data from the University of Kansas suggests that the gel-ripping or displacement mechanisms are more important in more permeable, strongly water-wet sandpacks. These findings help to explain why aqueous gels can reduce permeability to water more than to oil under different conditions. Since cement is the most commonly used material for water shutoff, we considered when gels are preferred over cements. Our analysis and experimental results indicated that cement cannot be expected to completely fill (top to bottom) a vertical fracture of any width, except near the wellbore. For vertical fractures with apertures less than 4 mm, the cement slurry will simply not penetrate very far into the fracture. For vertical fractures with apertures greater than 4 mm, the slurry may penetrate a substantial distance into the bottom part of the fracture. However, except near the wellbore, the upper part of the fracture will remain open due to gravity segregation. We compared various approaches to plugging fractures using gels, including (1) varying polymer content, (2) varying placement (extrusion) rate, (3) using partially formed gels, (4) using combinations of high and low molecular weight (Mw) polymers, (5) using secondary crosslinking reactions, (6) injecting un-hydrated polymer particles, and (7) incorporating particulates. All of these methods showed promise in some aspects, but required performance improvements in other aspects. All materials investigated to date showed significant performance variations with fracture width. High pressure gradients and limited distance of penetration are common problems in tight fractures. Gravity segregation and low resistance to breaching are common problems in wide fractures. These will be key issues to address in future work. Although gels can exhibit disproportionate permeability reduction in fractures, the levels of permeability reduction for oil flow are too high to allow practical exploitation in most circumstances. In contrast, disproportionate permeability reduction provided by gels that form in porous rock (adjacent to the fractures) has considerable potential in fractured systems.

  20. New resin gel for uranium determination by diffusive gradient in thin films technique.

    PubMed

    Gregusova, Michaela; Docekal, Bohumil

    2011-01-17

    A new resin gel based on Spheron-Oxin(®) chelating ion-exchanger with anchored 8-hydroxyquinoline functional groups was tested for application in diffusive gradient in thin film technique (DGT) for determination of uranium. Selectivity of uranium uptake from model carbonate loaded solutions of natural water was studied under laboratory conditions and compared with selectivity of the conventional Chelex 100 based resin gel. The affinity of Spheron-Oxin(®) functional groups enables determination of the overall uranium concentration in water containing carbonates up to the concentration level of 10(2) mg L(-1). The effect of uranium binding to the polyacrylamide (APA) and agarose diffusive gels (AGE) was also studied. Uranium is probably bound in both gels by a weak interaction with traces of acrylic acid groups in the structure of APA gel and with pyruvic and sulfonic acid groups in the AGE gel. These sorption effects can be eliminated to the negligible level by prolonged deployment of DGT probes or by disassembling probes after the 1-2 days post-sampling period that is sufficient for release of uranium from diffusive gel and its sorption in resin gel. PMID:21167996

  1. Volume transition in a gel driven by hydrogen bonding

    NASA Astrophysics Data System (ADS)

    Ilmain, Franck; Tanaka, Toyoichi; Kokufuta, Etsuo

    1991-01-01

    INTERACTIONS between macromolecules fall into four categories: ionic, hydrophobic, van der Waals and hydrogen bonding. Phase transitions in polymer gels provide a means of studying these interactions. Many gels will undergo reversible, discontinuous volume changes in response to changes in, for example, temperature, gel composition or light irradiation1-5. These transitions result from the competition between repulsive intermolecular forces, usually electrostatic in nature, that act to expand the polymer network, and an attractive force that acts to shrink it. Volume transitions in gels have been observed that are driven by all of the above-mentioned forces except hydrogen bonding (ref 6-10 T.T. et al, unpublished data; H. Inomata et al., personal communication). Here we report on a phase transition in an interpenetrating polymer network of poly(acrylamide) and poly(acrylic acid) that completes this picture-it is controlled by cooperative 'zipping' interactions between the molecules which result from hydrogen bonding. Cooperativity is an essential feature of the interactions, in that independent hydrogen bonds would not provide a sufficient driving force for the transition. A further novel characteristic of this phase transition is that the swelling (in water) is induced by an increase rather than a decrease in temperature.

  2. 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.

  3. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2004-03-01

    This technical progress report describes work performed from September 1, 2003, through February 29, 2004, for the project, ''Conformance Improvement Using Gels.'' We examined the properties of several ''partially formed'' gels that were formulated with a combination of high and low molecular weight HPAM polymers. After placement in 4-mm-wide fractures, these gels required about 25 psi/ft for brine to breach the gel (the best performance to date in fractures this wide). After this breach, stabilized residual resistance factors decreased significantly with increased flow rate. Also, residual resistance factors were up to 9 times greater for water than for oil. Nevertheless, permeability reduction factors were substantial for both water and oil flow. Gel with 2.5% chopped fiberglass effectively plugged 4-mm-wide fractures if a 0.5-mm-wide constriction was present. The ability to screen-out at a constriction appears crucial for particulate incorporation to be useful in plugging fractures. In addition to fiberglass, we examined incorporation of polypropylene fibers into gels. Once dispersed in brine or gelant, the polypropylene fibers exhibited the least gravity segregation of any particulate that we have tested to date. In fractures with widths of at least 2 mm, 24-hr-old gels (0.5% high molecular weight HPAM) with 0.5% fiber did not exhibit progressive plugging during placement and showed extrusion pressure gradients similar to those of gels without the fiber. The presence of the fiber roughly doubled the gel's resistance to first breach by brine flow. The breaching pressure gradients were not as large as for gels made with high and low molecular weight polymers (mentioned above). However, their material requirements and costs (i.e., polymer and/or particulate concentrations) were substantially lower than for those gels. A partially formed gel made with 0.5% HPAM did not enter a 0.052-mm-wide fracture when applying a pressure gradient of 65 psi/ft. This result suggests a lower limit of fracture width for entry of formed or partially formed gels (when reasonable pressure gradients are applied). In unfractured porous rock, we investigated the time dependence of oil and water permeabilities during various cycles of oil and water injection after placement of a Cr(III)-acetate-HPAM gel. Permeability to water stabilized rapidly (within 1 pore volume, PV), while permeability to oil stabilized gradually over the course of 100 PV. The behavior was surprisingly insensitive to core material (strongly water-wet Berea sandstone and strongly oil-wet porous polyethylene), core permeability (740 to 10,000 md), and applied pressure gradient (10 to 100 psi/ft).

  4. Modeling chemoresponsive polymer gels.

    PubMed

    Kuksenok, Olga; Deb, Debabrata; Dayal, Pratyush; Balazs, Anna C

    2014-01-01

    Stimuli-responsive gels are vital components in the next generation of smart devices, which can sense and dynamically respond to changes in the local environment and thereby exhibit more autonomous functionality. We describe recently developed computational methods for simulating the properties of such stimuli-responsive gels in the presence of optical, chemical, and thermal gradients. Using these models, we determine how to harness light to drive shape changes and directed motion in spirobenzopyran-containing gels. Focusing on oscillating gels undergoing the Belousov-Zhabotinksy reaction, we demonstrate that these materials can spontaneously form self-rotating assemblies, or pinwheels. Finally, we model temperature-sensitive gels that encompass chemically reactive filaments to optimize the performance of this system as a homeostatic device for regulating temperature. These studies could facilitate the development of soft robots that autonomously interconvert chemical and mechanical energy and thus perform vital functions without the continuous need of external power sources. PMID:24498954

  5. Long-term degradation of dilute polyacrylamide solutions in turbulent pipe flow

    SciTech Connect

    Choi, U.S.; Kasza, K.E.

    1989-05-01

    The long-term degradation behavior of 200 wppM polyacrylamide solution was studied experimentally in a closed recirculatory flow loop at temperatures of 7.2, 25 and 87.8/degree/C. The degradation behavior was found to be strongly dependent on temperature. The results indicate that, with flow shear similar to that encountered in practical DHC pipe flow, polyacrylamide solutions are highly effective and have a reasonable lifetime at chilled water temperature of 7.2/degree/C. 9 refs., 4 figs.

  6. Higher resolution microplate array diagonal gel electrophoresis: application to a multiallelic minisatellite.

    PubMed

    O'Dell, S D; Chen, X; Day, I N

    2000-01-01

    The 5' polymorphic region of the insulin (INS, MIM# 176730) gene contains a variable tandem repetition of 14-15 bp (a variable number of tandem repeats (VNTR) locus). After PCR amplification, we achieved precise sizing of class I alleles (range 641 to 843 bp) on 96-well open-face polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels, obtaining resolution of the 2% mobility difference which represents one tandem repeat. PCR products were run double-stranded, but no additional bands were generated except in the case of differences of three, two, and one repeat between alleles; none compromised allele identification, and in the latter case the heteroduplex was a useful confirmation signal. No end labelling of primers was required, as the sensitive Vistra Green intercalating dye for double strands was used for visualization of bands from diluted samples. Duracryl, a high mechanical-strength polyacrylamide derivative, proved to have good resolution properties for electrophoresis. A co-run ladder ensured precise binning without inter-lane variability. Simultaneous electrophoresis of gels in a thermostatically controlled tank allowed up to 1,000 samples to be run in 90 min. Gels were analyzed using a FluorImager 595 fluorescent scanning system, and alleles identified using a combination of Phoretix software for band migration measurement and Microsoft Excel to compute allele sizes. Unlike other systems for minisatellite allele sizing, throughput was not limited (in time or cost) by electrophoresis. PMID:10862086

  7. Native Knowledge in the Americas.

    ERIC Educational Resources Information Center

    Kidwell, Clara Sue

    1985-01-01

    Native American science is defined as activities of native peoples of the New World in observing physical phenomena and attempting to explain and control them. Problems in studying native science, ethnoscience and native science, archaeostronomy and ethnoastronomy, ethnobotany, agriculture, technology, and future directions are discussed. (JN)

  8. A gel probe equilibrium sampler for measuring arsenic porewater profiles and sorption gradients in sediments: I. Laboratory development

    USGS Publications Warehouse

    Campbell, K.M.; Root, R.; O'Day, P. A.; Hering, J.G.

    2008-01-01

    A gel probe equilibrium sampler has been developed to study arsenic (As) geochemistry and sorption behavior in sediment porewater. The gels consist of a hydrated polyacrylamide polymer, which has a 92% water content. Two types of gels were used in this study. Undoped (clear) gels were used to measure concentrations of As and other elements in sediment porewater. The polyacrylamide gel was also doped with hydrous ferric oxide (HFO), an amorphous iron (Fe) oxyhydroxide. When deployed in the field, HFO-doped gels introduce a fresh sorbent into the subsurface thus allowing assessment of in situ sorption. In this study, clear and HFO-doped gels were tested under laboratory conditions to constrain the gel behavior prior to field deployment. Both types of gels were allowed to equilibrate with solutions of varying composition and re-equilibrated in acid for analysis. Clear gels accurately measured solution concentrations (??1%), and As was completely recovered from HFO-doped gels (??4%). Arsenic speciation was determined in clear gels through chromatographic separation of the re-equilibrated solution. For comparison to speciation in solution, mixtures of As(III) and As(V) adsorbed on HFO embedded in gel were measured in situ using X-ray absorption spectroscopy (XAS). Sorption densities for As(III) and As(V) on HFO embedded in gel were obtained from sorption isotherms at pH 7.1. When As and phosphate were simultaneously equilibrated (in up to 50-fold excess of As) with HFO-doped gels, phosphate inhibited As sorption by up to 85% and had a stronger inhibitory effect on As(V) than As(III). Natural organic matter (>200 ppm) decreased As adsorption by up to 50%, and had similar effects on As(V) and As(III). The laboratory results provide a basis for interpreting results obtained by deploying the gel probe in the field and elucidating the mechanisms controlling As partitioning between solid and dissolved phases in the environment. ?? 2008 American Chemical Society.

  9. Toxicity of anionic polyacrylamide formulations when used for erosion control in agriculture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Addition of anionic polyacrylamide (PAM) to agricultural irrigation water can dramatically reduce erosion of soils. However, the toxicity of PAM to aquatic life, while often claimed to be low, has not been thoroughly evaluated. Five PAM formulations, including two oil-based products, one water-based...

  10. Comparison of Cationic and Unmodified Starches in Reactive Extrusion of Starch-Polyacrylamide Graft Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch and polyacrylamide (PAAm) were prepared using reactive extrusion in a corotating twin screw extruder. The effect of cationic starch modification was examined using unmodified and cationic dent starch (approximately 23% amylose) and waxy maize starch (approximately 2% amyl...

  11. Molecular Understanding and Structural-Based Design of Polyacrylamides and Polyacrylates as Antifouling Materials.

    PubMed

    Chen, Hong; Zhao, Chao; Zhang, Mingzhen; Chen, Qiang; Ma, Jie; Zheng, Jie

    2016-04-12

    Design and synthesis of highly bioinert and biocompatible antifouling materials are crucial for a broad range of biomedical and engineering applications. Among antifouling materials, polyacrylamides and polyacrylates have proved so promising because of cheap raw materials, ease of synthesis and applicability, and abundant functional groups. The strong surface hydration and the high surface packing density of polyacrylamides and polyacrylates are considered to be the key contributors to their antifouling property. In this article, we review our studies on the design and synthesis of a series of polyacrylamides and polyacrylates with different molecular structures. These polymers can be fabricated into different architectural forms (brushes, nanoparticles, nanogels, and hydrogels), all of which are highly resistant to the attachment of proteins, cells, and bacteria. We find that small structural changes in the polymers can lead to large enhancement in surface hydration and antifouling performance, both showing a positive correlation. This reveals a general design rule for effective antifouling materials. Furthermore, polyacrylamides and polyacrylates are readily functionalized with other bioactive compounds to achieve different new multifunctionalities. PMID:26986442

  12. USING POLYACRYLAMIDE TO CONTROL EROSION ON AGRICULTURAL AND DISTURBED SOILS IN RAINFED AREAS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Use of anionic polyacrylamide (PAM) as an erosion control soil amendment has been studied at the USDA-ARS National Soil Erosion Research Lab since the early 1990's. An initial field experiment in Indiana using simulated rainfall on a sloping silt loam soil found that 20 kg ha-1 of PAM could reduce s...

  13. Production of a polyacrylamide solution used in an oil recovery process

    SciTech Connect

    Luetzelschwab, W.E.

    1987-01-06

    A process is described for recovering oil from a subterranean oil-bearing formation having performance demands comprising the steps of: determining the performance demands of the formation; determining correlations between an initial polymerization reaction parameter of initiator level and partially hydrolyzed polyacrylamide solution properties of screen factor and viscosity, each correlation having a discontinuity; selecting a value of the initiator level below each discontinuity such that the selected value of the initiator level is capable of producing a partially hydrolyzed polyacrylamide solution having values of the properties of viscosity and screen factor relatively sensitive to varying the initiator level and capable of meeting the performance demands of the formation; producing the partially hydrolyzed polyacrylamide solution having the values of the properties relatively sensitive to varying the initiator level and capable of meeting the performance demands by polymerizing an acrylamide monomer using a polymerization initiator at the selected value; and injecting the partially hydrolyzed polyacrylamide solution into the formation to improve oil recovery therefrom.

  14. Polyacrylamide and biopolymer effects on flocculation, aggregate stability, and water seepage in a silt loam

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Researcher’s seek a more renewable and natural alternative for water soluble anionic polyacrylamide (PAM), a highly-effective, petroleum-derived polymer used in agriculture to control erosion and reduce water seepage from unlined irrigation structures. This study evaluated two anionic polymers: a ba...

  15. Polyacrylamide Molecular Weight and Phosphogypsum Effects on Infiltration and Erosion in Semi-Arid Soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seal formation at the surface of semi-arid soils during rainstorms reduces soil infiltration rate (IR) and causes runoff and erosion. Surface application of dry anionic polyacrylamide (PAM) with high molecular weight (MW) has been found to be effective in stabilizing soil aggregates, and decreasing ...

  16. Reduction of Interrill Erosion by different application methods of Polyacrylamide and Gypsum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil erosion has been studied from different perspectives. This paper presents results on interrill erosion for three different application methods of Polyacrylamide (PAM) and Gypsum. Small interrill plots (0.74 m2) were packed with a highly erodible sieved soil and surface was prepared for it to ha...

  17. Aggregate stability as affected by polyacrylamide molecular weight, soil texture and water quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The favorable effects of the environmentally friendly, non toxic, anionic polyacrylamide (PAM) as a soil conditioner have long been established. However, some uncertainties exist regarding the effects of PAM molecular weight (MW) on its performance as a soil amendment and the ability of PAM to penet...

  18. Polyacrylamide molecular weight and phosphogypsum effects on infiltration and erosion in semi-arid soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seal formation at the surface of semi-arid soils during rainstorms reduces soil infiltration rate (IR) and causes runoff and erosion. Surface application of dry anionic polyacrylamide (PAM) with high molecular weight (MW) has been found to be effective in stabilizing soil aggregates, and decreasing ...

  19. Carbon and nitrogen stable isotope ratios can estimate anionic polyacrylamide degradation in soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Water soluble anionic polyacrylamide (PAM) is a highly effective erosion-preventing and infiltration-enhancing polymer, when applied at rates of 1 to 10 g/m-3 in furrow irrigation water. PAM degradation has not directly been measured in soil or water. Natural abundance of the carbon (13C/12C) isoto...

  20. Application of Polyacrylamide to Reduce Phosphorus Losses from Chinese Purple Soil: A Laboratory and Field Investigation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The application of anionic polyacrylamide (PAM) for the control of phosphorus (P) losses from a steeply sloping Chinese purple soil was studied in both a laboratory soil column experiment and a field experiment. The results showed that PAM has an important inhibitory impact on vertical P transport i...

  1. Effects of Polyacrylamide and Organic Matter on Microbes associated to Soil Aggregation of Norfolk Loamy Sand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyacrylamide (PAM, anionic formulation of molecular size 12 MDa and 35% charge density) has been reported to increase aggregation and improve soil physical properties in United States southeastern Coastal Plain loamy sand soils, but nothing is known about the effects of PAM on microbes associated ...

  2. Erosion control practices integrated with polyacrylamide for nutrient reduction in rill irrigation runoff

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this research was to assess soil conservation practices for improving water quality of return flows from rill irrigation in the Yakima River Basin, Washington, by combining patch application of polyacrylamide (PAM) with an additional erosion control practice. A two-year field study ...

  3. Effects of Polyacrylamide Molecular Weight, Soil Texture and Electrolyte Concentration on Drainable Porosity and Aggregate Stability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The literature reports on the intricate relations between soil type and molecular weight (MW) of polyacrylamide (PAM) with respect to PAM efficacy as a soil conditioner. This relation may depend on the ability of PAM to penetrate into aggregates and thus stabilize both outer and inner aggregate surf...

  4. Polyacrylamide effects on aggregate and structure stability of soils with different clay mineralogy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adding anionic polyacrylamide (PAM) to soils stabilizes existing aggregates and improves bonding between and aggregation of soil particles. However, the dependence of PAM efficacy as an aggregate stabilizing agent with soils having different clay mineralogy has not been studied. Sixteen soil samples...

  5. Polyacrylamide treatments for reducing seepage in soil-lined reservoirs: A field evaluation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Irrigation water supplies are becoming limited and there is a need to extend the usefulness of current water resources. Previous laboratory studies demonstrated that certain water soluble polyacrylamide solution (WSPAM) and cross-linked PAM granule (XPAM) treatments effectively reduced infiltration...

  6. PECTIN AND POLYACRYLAMIDE COMPOSITE HYDROGELS: EFFECT OF PECTIN ON STRUCTURAL AND DYNAMIC MECHANICAL PROPERTIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Composite hydrogels of pectin and polyacrylamide were synthesized and evaluated by scanning electron microscopy, atomic force microscopy, light microscopy, and by dynamic mechanical analysis. The cross-linking polymerization of acrylamide in pectin solution resulted in a composite having a macropo...

  7. POLYACRYLAMIDE (PAM) APPLICATION EFFECTIVENESS IN REDUCING SOIL EROSION FROM SUGARCANE FIELDS IN SOUTHERN LOUISIANA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A field experiment was conducted in the spring of 2002 to determine the effectiveness of high molecular weight anionic polyacrylamide (PAM) in stabilizing quarter drains(reducing soil erosion) on fields planted to sugarcane. Quarter drains are small surface ditches perpendicular to sugarcane furrow...

  8. POLYACRYLAMIDE EFFECTS ON INFILTRATION IN SAN JOAQUIN VALLEY SANDY LOAM SOILS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low infiltration rates constrain effective and economical irrigation on some sandy loam soils on the east side of California's San Joaquin Valley. Polyacrylamide (PAM) has increased soil infiltration in other areas of the U.S., especially where soil erosion is a problem. We applied low concentrati...

  9. AN EVALUATION OF POLYACRYLAMIDE AND SURFACE HYDROLOGIC EFFECTS ON RUNOFF WATER QUALITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyacrylamide (PAM) and surface hydrologic condition are known to affect sediment and runoff water quality as individual factors, but their combined effects have not been quantified. In this study, we quantified the interactive effects of PAM and three near-surface hydrologic conditions: drainage,...

  10. Soil aggregate stability as affected by clay mineralogy and polyacrylamide addition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The addition of polyacrylamide (PAM) to soil leads to stabilization of existing aggregates and improved bonding between, and aggregation of adjacent soil particles However, the dependence of PAM efficacy as an aggregate stabilizing agent on soil-clay mineralogy has not been studied. Sixteen soil sam...

  11. Gel for Simultaneous Chemical Imaging of Anionic and Cationic Solutes Using Diffusive Gradients in Thin Films

    PubMed Central

    2013-01-01

    We report on a novel gel based on diffusive gradients in thin films (DGT) for the simultaneous measurement of cations and anions and its suitability for high resolution chemical imaging by using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The new high resolution mixed binding gel (HR-MBG) is based on zirconium-hydroxide and suspended particulate reagent-iminodiacetate (SPR-IDA) as resin materials which are embedded in an ether-based urethane polymer hydrogel. The use of this polymer hydrogel material allows the production of ultrathin, highly stable and tear-proof resin gel layers with superior handling properties compared to existing ultrathin polyacrylamide gels. The gel was characterized regarding its uptake kinetics, the anion and cation capacities, and the effects of pH, ionic strength, and aging on the performance of the HR-MBG. Our results demonstrate the capability of this novel gel for concomitant sampling of anions and cations. The suitability of this new gel type for DGT chemical imaging at submm spatial resolution in soils using LA-ICPMS is shown. 2D images of P, As, Co, Cu, Mn, and Zn distributions around roots of Zea mays L. demonstrate the new opportunities offered by the HR-MBG for high-resolution mapping of solute dynamics in soil and sediment hotspots, such as the rhizosphere, by simultaneous observation of anionic and cationic solute species. PMID:24256092

  12. Gel for simultaneous chemical imaging of anionic and cationic solutes using diffusive gradients in thin films.

    PubMed

    Kreuzeder, Andreas; Santner, Jakob; Prohaska, Thomas; Wenzel, Walter W

    2013-12-17

    We report on a novel gel based on diffusive gradients in thin films (DGT) for the simultaneous measurement of cations and anions and its suitability for high resolution chemical imaging by using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The new high resolution mixed binding gel (HR-MBG) is based on zirconium-hydroxide and suspended particulate reagent-iminodiacetate (SPR-IDA) as resin materials which are embedded in an ether-based urethane polymer hydrogel. The use of this polymer hydrogel material allows the production of ultrathin, highly stable and tear-proof resin gel layers with superior handling properties compared to existing ultrathin polyacrylamide gels. The gel was characterized regarding its uptake kinetics, the anion and cation capacities, and the effects of pH, ionic strength, and aging on the performance of the HR-MBG. Our results demonstrate the capability of this novel gel for concomitant sampling of anions and cations. The suitability of this new gel type for DGT chemical imaging at submm spatial resolution in soils using LA-ICPMS is shown. 2D images of P, As, Co, Cu, Mn, and Zn distributions around roots of Zea mays L. demonstrate the new opportunities offered by the HR-MBG for high-resolution mapping of solute dynamics in soil and sediment hotspots, such as the rhizosphere, by simultaneous observation of anionic and cationic solute species. PMID:24256092

  13. In-phantom dosimetry for BNCT with Fricke and normoxic-polymer gels

    NASA Astrophysics Data System (ADS)

    Gambarini, G.; Agosteo, S.; Carrara, M.; Gay, S.; Mariani, M.; Pirola, L.; Vanossi, E.

    2006-05-01

    Measurements of in-phantom dose distributions and images are important for Boron Neutron Capture Therapy treatment planning. The method for spatial determination of absorbed doses in thermal or epithermal neutron fields, based on Fricke-xylenol-orange-infused gel dosimeters in form of layers, has revealed to be very reliable, as gel layer dosimeters give the possibility of obtaining spatial dose distributions and measurements of each dose contribution in neutron fields, by means of a properly studied procedure. Quite recently, BNCT has been applied to treat liver metastases; in this work the results of in-phantom dosimetry for explanted liver in BNCT treatments are described. Moreover, polyacrylamide gel (PAG) dosimeters in which a polymerization process appears as a consequence of absorbed dose, have been recently tested, because of their characteristic absence of diffusion. In fact, due to the diffusion of ferric ions, Fricke-gel dosimeters require prompt analysis after exposure to avoid spatial information loss. In this work the preliminary results of a study about the reliability of polymer gel in BNCT dosimetry are also discussed. Gel layers have been irradiated in a phantom exposed in the thermal column of the TRIGA MARK II reactor (Pavia). The results obtained with the two kinds of gel dosimeter have been compared.

  14. A Pneumatic Device for Rapid Loading of DNA Sequencing Gels

    PubMed Central

    Panussis, Dimitrios A.; Cook, Mark W.; Rifkin, Lisa L.; Snider, Jacqueline E.; Strong, Joseph T.; McGrane, Rebecca M.; Wilson, Richard K.; Mardis, Elaine R.

    1998-01-01

    This work describes the design and construction of a device that facilitates the loading of DNA samples onto polyacrylamide gels for detection in the Perkin Elmer/Applied Biosystems (PE/ABI) 373 and 377 DNA sequencing instruments. The device is mounted onto the existing gel cassettes and makes the process of loading high-density gels less cumbersome while the associated time and errors are reduced. The principle of operation includes the simultaneous transfer of the entire batch of samples, in which a spring-loaded air cylinder generates positive pressure and flexible silica capillaries transfer the samples. A retractable capillary array carrier allows the delivery ends of the capillaries to be held up clear of the gel during loader attachment on the gel plates, while enabling their insertion in the gel wells once the device is securely mounted. Gel-loading devices capable of simultaneously transferring 72 samples onto the PE/ABI 373 and 377 are currently being used in our production sequencing groups while a 96-sample transfer prototype undergoes testing. PMID:9582197

  15. Synthesis of p -and n-type Gels Doped with Ionic Charge Carriers

    NASA Astrophysics Data System (ADS)

    Alveroglu, E.; Yilmaz, Y.

    2010-03-01

    In this study, we synthesized the new kinds of semiconducting polymeric gels having negative ( n-type) and positive ( p-type) counter ions as charge carriers. The polyacrylamide gel was doped with pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt), having {text{SO}}3^{ - } ions as side groups and Na+ as counter ions, so-called p-type semiconducting gel. The doping process was performed during the polymerization where the pyranine binds to the polymer strands over OH group chemically via radical addition. In a similar way, N-isopropylacrylamide (NIPA) gel was doped with methacrylamidopropyltrimethyl ammonium chloride (MAPTAC), having Cl- as counter ions, so-called n-type semiconducting gel. Here MAPTAC was embedded by copolymerization within the polymer network (NIPA). These semiconducting gels can show different electrical properties by changing the concentration of the doping agents, swelling ratio etc. We have shown that the pn junction, formed by combining p-type and n-type gels together in close contact, rectifies the current similar to the conventional Si and Ge diodes.

  16. Saos-2 cell-mediated mineralization on collagen gels: Effect of densification and bioglass incorporation.

    PubMed

    Liu, Gengbo; Pastakia, Meet; Fenn, Michael B; Kishore, Vipuil

    2016-05-01

    Plastic compression is a collagen densification process that has been widely used for the development of mechanically robust collagen-based materials. Incorporation of bioglass within plastically compressed collagen gels has been shown to mimic the microstructural properties of native bone and enhance in vitro cell-mediated mineralization. The current study seeks to decouple the effects of collagen densification and bioglass incorporation to understand the interplay between collagen packing density and presence of bioglass on cell-mediated mineralization. Saos-2 cell-mediated mineralization was assessed as a measure of the osteoconductivity of four different collagen gels: (1) uncompressed collagen gel (UC), (2) bioglass incorporated uncompressed collagen gel (UC + BG), (3) plastically compressed collagen gel (PC), and (4) bioglass incorporated plastically compressed collagen gel (PC + BG). The results indicated that collagen densification enhanced mineralization as shown by SEM, increased alkaline phosphatase activity and produced significantly higher amounts of mineralized nodules on PC gels compared to UC gels. Further, the amount of nodule formation on PC gels was significantly higher compared to UC + BG gels indicating that increase in matrix stiffness due to collagen densification had a greater effect on cell-mediated mineralization compared to bioglass incorporation into loosely packed UC gels. Incorporation of bioglass into PC gels further enhanced mineralization as evidenced by significantly larger nodule size and higher amount of mineralization on PC + BG gels compared to PC gels. In conclusion, collagen densification via plastic compression improves the osteoconductivity of collagen gels. Further, incorporation of bioglass within PC gels has an additive effect and further enhances the osteoconductivity of collagen gels. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1121-1134, 2016. PMID:26750473

  17. Microfluidics with Gel Emulsions

    NASA Astrophysics Data System (ADS)

    Priest, Craig; Surenjav, Enkhtuul; Herminghaus, Stephan; Seemann, Ralf

    2006-03-01

    Microfluidic processing is usually achieved using single phase liquids. Instead, we use monodisperse emulsions to compartment liquids within microchannel geometries. At low continuous phase volume fractions, droplets self-organize to form well-defined arrangements, analogous to foam. While it is well-known that confined geometries can induce rearrangement of foam compartments at the millimeter-scale, similar dynamics are also expected for gel emulsions. We have studied online generation, organization and manipulation of gel emulsions using a variety of microchannel geometries. ``Passive'' reorganization, based on fixed channel geometries, can be supplemented by ``active'' manipulation by incorporating a ferrofluid phase. A ferromagnetic phase facilitates reorganization of liquid compartments on demand using an electromagnetic trigger. Moreover, coalescence between adjacent compartments within a gel emulsion can be induced using electrical potential. Microfluidics using gel emulsions will be well-suited for combinatorial chemistry, DNA sequencing, drug screening and protein crystallizations.

  18. Effect of the exothermal polymerization reaction on polymer gel dosimetric measurements

    NASA Astrophysics Data System (ADS)

    Sedaghat, Mahbod; Bujold, Rachel; Lepage, Martin

    2010-11-01

    Discrepancies in polymer gel dosimetric measurements have been observed between containers of different sizes receiving the same radiation dose. We hypothesized that these deviations are caused by a change in the rate of polymerization due to internal heat increase in the gel containers resulting from the exothermic polymerization of monomers. Here, we test this hypothesis in a polyacrylamide gel dosimeter by recording the temperature in glass phantoms of different sizes during and after irradiation. The dose response of the samples was determined with magnetic resonance imaging. The difference of R2 values along the depth of the containers was below ±1%. We discuss that this small difference can be attributed to variations in the rate of gelatin cooling during manufacture rather than to the measured heat increase during irradiation.

  19. Detection and characterization of heparin-binding proteins with a gel overlay procedure

    SciTech Connect

    Mehlman, T.; Burgess, W.H. )

    1990-07-01

    The binding of {sup 125}I-labeled derivatives of heparin has been used by several investigators to identify heparin-binding fragments of different heparin-binding proteins. In this report we utilize the procedure described by J.W. Smith and D.J. Knauer (1987, Anal. Biochem. 160, 105-114) to produce {sup 125}I-fluorescein-heparin. Using this derivative, we compare the use of gel overlay procedures with Western blot procedures for the detection of heparin-binding proteins following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We show that the gel overlay procedure is a relatively simple and sensitive method for visualizing heparin-binding proteins. In addition, we use the procedure to characterize the heparin-binding properties of heparin-binding growth factor 1 (acidic fibroblast growth factor) with synthetic peptide competitors and site-directed mutants of the growth factor.

  20. Janus Gel Fabrication Using Liquid Drop Coalescence and Limited Mixing in the Hele-Shaw Geometry

    NASA Astrophysics Data System (ADS)

    Gonzalez, Brittany; Moran, Alexis; Lee, Donghee; Ryu, Sangjin

    2015-11-01

    Hydrogel substrates of tunable stiffness have been actively utilized for in vitro cell mechanobiology study. Here we present a new method to fabricate Janus polyacrylamide gel based on limited mixing between liquid drops coalescing in the Hele-Shaw geometry. Two pre-polymer drops with different concentrations were sandwiched and squeezed between two parallel glass surfaces. Once the drops coalesced in the decreased gap between the surfaces, gelation was initiated by UV light exposure with various time delays. AFM nano-indentation was utilized to map the Young's modulus of obtained gels. Fabricated Janus gels had two regions of different Young's moduli interfaced by the stiffness gradient zone, and the width of the gradient zone increased with the delay time. We acknowledge support from Bioengineering for Human Health grant from UNL and UNMC, and NSF REU grant for UNL.

  1. Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility.

    PubMed

    Jin, Li-Tai; Li, Xiao-Kun; Cong, Wei-Tao; Hwang, Sun-Young; Choi, Jung-Kap

    2008-12-15

    A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band. PMID:18804088

  2. Crystallization from Gels

    NASA Astrophysics Data System (ADS)

    Narayana Kalkura, S.; Natarajan, Subramanian

    Among the various crystallization techniques, crystallization in gels has found wide applications in the fields of biomineralization and macromolecular crystallization in addition to crystallizing materials having nonlinear optical, ferroelectric, ferromagnetic, and other properties. Furthermore, by using this method it is possible to grow single crystals with very high perfection that are difficult to grow by other techniques. The gel method of crystallization provides an ideal technique to study crystal deposition diseases, which could lead to better understanding of their etiology. This chapter focuses on crystallization in gels of compounds that are responsible for crystal deposition diseases. The introduction is followed by a description of the various gels used, the mechanism of gelling, and the fascinating phenomenon of Liesegang ring formation, along with various gel growth techniques. The importance and scope of study on crystal deposition diseases and the need for crystal growth experiments using gel media are stressed. The various crystal deposition diseases, viz. (1) urolithiasis, (2) gout or arthritis, (3) cholelithiasis and atherosclerosis, and (4) pancreatitis and details regarding the constituents of the crystal deposits responsible for the pathological mineralization are discussed. Brief accounts of the theories of the formation of urinary stones and gallstones and the role of trace elements in urinary stone formation are also given. The crystallization in gels of (1) the urinary stone constituents, viz. calcium oxalate, calcium phosphates, uric acid, cystine, etc., (2) the constituents of the gallstones, viz. cholesterol, calcium carbonate, etc., (3) the major constituent of the pancreatic calculi, viz., calcium carbonate, and (4) cholic acid, a steroidal hormone are presented. The effect of various organic and inorganic ions, trace elements, and extracts from cereals, herbs, and fruits on the crystallization of major urinary stone and gallstone constituents are described. In addition, tables of gel-grown organic and inorganic crystals are provided.

  3. Fluorescent staining of gels.

    PubMed

    Buxbaum, Engelbert

    2012-01-01

    Certain transition metal complexes show intensive fluorescence when bound to proteins. They can be used to stain gels after electrophoresis with a sensitivity approaching that of silver staining, but in a much simpler and more reproducible procedure. Stains can be prepared easily and at a fraction of the cost of commercially available reagents.Hydrophobic dyes can be used to stain gels without fixing; they do not interfere with later blotting or electro-elution. PMID:22585519

  4. Conformance Improvement Using Gels

    SciTech Connect

    Seright, Randall S.; Schrader; II Hagstrom, John; Wang, Ying; Al-Dahfeeri, Abdullah; Marin, Amaury

    2002-09-26

    This research project had two objectives. The first objective was to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective was to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil.

  5. Native plant diversity increases herbivory to non-natives

    PubMed Central

    Pearse, Ian S.; Hipp, Andrew L.

    2014-01-01

    There is often an inverse relationship between the diversity of a plant community and the invasibility of that community by non-native plants. Native herbivores that colonize novel plants may contribute to diversity–invasibility relationships by limiting the relative success of non-native plants. Here, we show that, in large collections of non-native oak trees at sites across the USA, non-native oaks introduced to regions with greater oak species richness accumulated greater leaf damage than in regions with low oak richness. Underlying this trend was the ability of herbivores to exploit non-native plants that were close relatives to their native host. In diverse oak communities, non-native trees were on average more closely related to native trees and received greater leaf damage than those in depauperate oak communities. Because insect herbivores colonize non-native plants that are similar to their native hosts, in communities with greater native plant diversity, non-natives experience greater herbivory. PMID:25232143

  6. Native plant diversity increases herbivory to non-natives.

    PubMed

    Pearse, Ian S; Hipp, Andrew L

    2014-11-01

    There is often an inverse relationship between the diversity of a plant community and the invasibility of that community by non-native plants. Native herbivores that colonize novel plants may contribute to diversity-invasibility relationships by limiting the relative success of non-native plants. Here, we show that, in large collections of non-native oak trees at sites across the USA, non-native oaks introduced to regions with greater oak species richness accumulated greater leaf damage than in regions with low oak richness. Underlying this trend was the ability of herbivores to exploit non-native plants that were close relatives to their native host. In diverse oak communities, non-native trees were on average more closely related to native trees and received greater leaf damage than those in depauperate oak communities. Because insect herbivores colonize non-native plants that are similar to their native hosts, in communities with greater native plant diversity, non-natives experience greater herbivory. PMID:25232143

  7. Native Americans: Subject Guide.

    ERIC Educational Resources Information Center

    Bonanni, Mimmo; Etter, Patricia A.

    This annotated subject guide lists reference material that deals with Native Americans and is available in the Arizona State University Libraries. Entries were published 1933-98, but mostly in the 1980s-90s. The guide is not comprehensive, but rather a selective list of resources useful for researching a topic in a variety of fields. The guide

  8. Native Indian Leadership.

    ERIC Educational Resources Information Center

    Jules, Felicity

    1988-01-01

    Identifies valued qualities and behaviors of Indian leaders through a literature review and unstructured interviews with three British Columbian tribal elders. Develops a model of Native leadership emphasizing connection to the people, wisdom, humility, personal integrity, service orientation, and the facilitator role. Contains 22 references. (SV)

  9. Native American Women.

    ERIC Educational Resources Information Center

    Green, Rayna

    1980-01-01

    Reviews trends in the literature on Native American women and notes significant changes in recent years. Suggests that scholarly issues may have to be subordinated to community needs, and that feminist scholars should direct their studies to those issues most important to the group being studied. (ST)

  10. Rebuilding Native American Communities

    ERIC Educational Resources Information Center

    Coyhis, Don; Simonelli, Richard

    2005-01-01

    The Wellbriety Movement in Native American communities draws on the wisdom and participation of traditional elders. Beginning with a basic community teaching called the Four Laws of Change and the Healing Forest Model, the Wellbriety Movement blends Medicine Wheel knowledge with the 12 Steps of Alcoholics Anonymous to provide culture-specific…

  11. Native American Literature.

    ERIC Educational Resources Information Center

    Porter, C. Fayne; And Others

    Designed to accommodate a semester course in Native American Literature for secondary students, this teacher's guide includes a general introduction, a statement of the philosophy and goals upon which it is predicated, a nine-week block on post-Columbian literature, a nine-week block on oral literature, separate appendices for each block, a…

  12. Native Westslope Cutthroat Trout

    Native westslope cutthroat trout swim in the north fork of the Flathead River in northwestern Montana. The region is recognized as a range-wide stronghold for genetically pure westslope cutthroat trout. However, rainbow trout invasion and hybridization threatens these p...

  13. Native Westslope Cutthroat Trout

    Native westslope cutthroat trout swim in the north fork of the Flathead River in northwestern Montana. This region is recognized as a range-wide stronghold for genetically pure westslope cutthroat trout. However, rainbow trout invasion and hybridization threatens these ...

  14. Native American Resource Book.

    ERIC Educational Resources Information Center

    Spears, Carl D., Comp.; And Others

    Focusing on the Southeastern American Indian cultures, this Native American resource guide is designed for use in the elementary and secondary schools of the East Baton Rouge Parish and is a product of a 1975 Indian Advisory Committee composed of Indian parents, teachers, and staff members. Objectives of these materials require the Indian student,…

  15. Native American Cultural Groups.

    ERIC Educational Resources Information Center

    Roy, Loriene, Comp.

    Part of a larger report on the Four Directions Project, an American Indian technology innovation project, this section includes 13 "pathfinders" to locating information on Native American and other indigenous cultural groups. The pathfinders were designed by students in the Graduate School of Library and Information Science at the University of…

  16. Native American Perspectives.

    ERIC Educational Resources Information Center

    Smith, Walter S.

    1998-01-01

    On the Fajada Butte in New Mexico, 11th-century Anasazi constructed a site that marks the high and low points of the orbits of the sun and the moon. This unit on astronomy challenges students to think differently about the moon and about the ability of native people to understand the natural world. Includes resources for further study. (PVD)

  17. Symbolicity Among Native Americans.

    ERIC Educational Resources Information Center

    Hill, L. Brooks; Lujan, Philip

    Within the framework of "symbolicity" and "nativistic movement" the paper presents a "reasonably balanced and illustrative" examination of selected negative and positive trends in Native American symbolicity. Symbolicity is defined as the state, condition, and tendency of people to organize their perceptions and experience into symbols and symbol…

  18. Rebuilding Native American Communities

    ERIC Educational Resources Information Center

    Coyhis, Don; Simonelli, Richard

    2005-01-01

    The Wellbriety Movement in Native American communities draws on the wisdom and participation of traditional elders. Beginning with a basic community teaching called the Four Laws of Change and the Healing Forest Model, the Wellbriety Movement blends Medicine Wheel knowledge with the 12 Steps of Alcoholics Anonymous to provide culture-specific

  19. Exploring Native American Symbolism.

    ERIC Educational Resources Information Center

    Dufrene, Phoebe

    This paper described the events and results of a workshop on Native American symbolism presented to educators and held in Kansas City, Missouri. The presenter maintained that some of the most crucial problems facing U.S. educators and students are caused by racial misunderstandings, and that the universality of artistic expression can be a vehicle…

  20. The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc.

    PubMed

    Shu, Cindy; Hughes, Clare; Smith, Susan M; Smith, Margaret M; Hayes, Anthony; Caterson, Bruce; Little, Christopher B; Melrose, James

    2013-10-01

    Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P < 0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations. PMID:23756834

  1. NATIVE HEALTH DATABASES: NATIVE HEALTH RESEARCH DATABASE (NHRD)

    EPA Science Inventory

    The Native Health Databases contain bibliographic information and abstracts of health-related articles, reports, surveys, and other resource documents pertaining to the health and health care of American Indians, Alaska Natives, and Canadian First Nations. The databases provide i...

  2. NATIVE HEALTH DATABASES: NATIVE HEALTH HISTORY DATABASE (NHHD)

    EPA Science Inventory

    The Native Health Databases contain bibliographic information and abstracts of health-related articles, reports, surveys, and other resource documents pertaining to the health and health care of American Indians, Alaska Natives, and Canadian First Nations. The databases provide i...

  3. Studying Native America: Problems and Prospects.

    ERIC Educational Resources Information Center

    Thornton, Russell, Ed.

    Based on a conference, this volume examines the past, present, and future of Native American studies. Native American studies seeks to understand Native Americans, America, and the world from a Native American indigenous perspective, and thereby broaden the education of both Native and non-Native Americans. Part 1 asks who Native Americans are

  4. Studying Native America: Problems and Prospects.

    ERIC Educational Resources Information Center

    Thornton, Russell, Ed.

    Based on a conference, this volume examines the past, present, and future of Native American studies. Native American studies seeks to understand Native Americans, America, and the world from a Native American indigenous perspective, and thereby broaden the education of both Native and non-Native Americans. Part 1 asks who Native Americans are…

  5. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2002-02-28

    This technical progress report describes work performed from June 20 through December 19, 2001, for the project, ''Conformance Improvement Using Gels''. Interest has increased in some new polymeric products that purport to substantially reduce permeability to water while causing minimum permeability reduction to oil. In view of this interest, we are currently studying BJ's Aqua Con. Results from six corefloods revealed that the Aqua Con gelant consistently reduced permeability to water more than that to oil. However, the magnitude of the disproportionate permeability reduction varied significantly for the various experiments. Thus, as with most materials tested to date, the issue of reproducibility and control of the disproportionate permeability remains to be resolved. Concern exists about the ability of gels to resist washout after placement in fractures. We examined whether a width constriction in the middle of a fracture would cause different gel washout behavior upstream versus downstream of the constriction. Tests were performed using a formed Cr(III)-acetate-HPAM gel in a 48-in.-long fracture with three sections of equal length, but with widths of 0.08-, 0.02-, and 0.08-in., respectively. The pressure gradients during gel extrusion (i.e., placement) were similar in the two 0.08-in.-wide fracture sections, even though they were separated by a 0.02-in.-wide fracture section. The constriction associated with the middle fracture section may have inhibited gel washout during the first pulse of brine injection after gel placement. However, during subsequent phases of brine injection, the constriction did not inhibit washout in the upstream fracture section any more than in the downstream section.

  6. Native Peoples-Native Homelands Climate Change Workshop: Lessons Learned

    NASA Technical Reports Server (NTRS)

    Maynard, Nancy G.

    2003-01-01

    The Native Peoples-Native Homelands Climate Change Workshop was held on October 28 through November 01,1998, as part of a series of workshops being held around the U.S. to improve the understanding of the potential consequences of climate variability and change for the Nation. This workshop was specifically designed by Native Peoples to examine the impacts of climate change and extreme weather variability on Native Peoples and Native Homelands from an indigenous cultural and spiritual perspective and to develop recommendations as well as identify potential response actions. The workshop brought together interested Native Peoples, representatives of Tribal governments, traditional elders, Tribal leaders, natural resource managers, Tribal College faculty and students, and climate scientists fiom government agencies and universities. It is clear that Tribal colleges and universities play a unique and critical role in the success of these emerging partnerships for decision-making in addition to the important education function for both Native and non-Native communities such as serving as a culturally-appropriate vehicle for access, analysis, control, and protection of indigenous cultural and intellectual property. During the discussions between scientists and policy-makers from both Native and non-Native communities, a number of important lessons emerged which are key to building more effective partnerships between Native and non-Native communities for collaboration and decision-making for a more sustainable future. This talk summarizes the key issues, recommendations, and lessons learned during this workshop.

  7. Native Peoples-Native Homelands Climate Change Workshop: Lessons Learned

    NASA Astrophysics Data System (ADS)

    Maynard, N. G.

    2003-12-01

    The Native Peoples-Native Homelands Climate Change Workshop was held on October 28 through November 01, 1998, as part of a series of workshops being held around the U.S. to improve the understanding of the potential consequences of climate variability and change for the Nation. This workshop was specifically designed by Native Peoples to examine the impacts of climate change and extreme weather variability on Native Peoples and Native Homelands from an indigenous cultural and spiritual perspective and to develop recommendations as well as identify potential response actions. The workshop brought together interested Native Peoples, representatives of Tribal governments, traditional elders, Tribal leaders, natural resource managers, Tribal College faculty and students, and climate scientists from government agencies and universities. It is clear that Tribal colleges and universities play a unique and critical role in the success of these emerging partnerships for decision-making in addition to the important education function for both Native and non-Native communities such as serving as a culturally- appropriate vehicle for access, analysis, control, and protection of indigenous cultural and intellectual property. During the discussions between scientists and policy-makers from both Native and non-Native communities, a number of important lessons emerged which are key to building more effective partnerships between Native and non-Native communities for collaboration and decision-making for a more sustainable future. This talk summarizes the key issues, recommendations, and lessons learned during this workshop.

  8. Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis

    PubMed Central

    Lee, Andrew; Whitesides, George M.

    2010-01-01

    This paper describes the development of a method that uses Capillary Gel Electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((Pi)n). Resolution of (Pi)n on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV-absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (Pi)n with n up to ~70; preparation and use of authentic standards enables the identification of peaks for (Pi)n with n = 1 - 10. The main advantages of this method over previously reported methods for analyzing mixtures of (Pi)n (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (Pi)n by suppressed conductivity. The method is useful for analyzing (Pi)n generated by the dehydration of Pi at low temperature (125 - 140 °C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters). PMID:20704373

  9. 76 FR 22413 - Alaska Native Claims Selection

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-21

    ...) to Afognak Native Corporation, Successor in Interest to Port Lions Native Corporation. The decision... surface estate is conveyed to Afognak Native Corporation, Successor in Interest to Port Lions...

  10. Digital Natives or Digital Tribes?

    ERIC Educational Resources Information Center

    Watson, Ian Robert

    2013-01-01

    This research builds upon the discourse surrounding digital natives. A literature review into the digital native phenomena was undertaken and found that researchers are beginning to identify the digital native as not one cohesive group but of individuals influenced by other factors. Primary research by means of questionnaire survey of technologies…

  11. Readings in Canadian Native Studies.

    ERIC Educational Resources Information Center

    Price, John A.

    1986-01-01

    After noting that Canadian and American scholars virtually ignore each other in the field of Native Studies, this paper summarizes sources of information for anyone interested in Canadian Native studies. The introduction mentions university programs and departments, newspaper coverage of Native issues, number of books and monographs published per…

  12. Spatially resolved multicomponent gels

    NASA Astrophysics Data System (ADS)

    Draper, Emily R.; Eden, Edward G. B.; McDonald, Tom O.; Adams, Dave J.

    2015-10-01

    Multicomponent supramolecular systems could be used to prepare exciting new functional materials, but it is often challenging to control the assembly across multiple length scales. Here we report a simple approach to forming patterned, spatially resolved multicomponent supramolecular hydrogels. A multicomponent gel is first formed from two low-molecular-weight gelators and consists of two types of fibre, each formed by only one gelator. One type of fibre in this ‘self-sorted network’ is then removed selectively by a light-triggered gel-to-sol transition. We show that the remaining network has the same mechanical properties as it would have done if it initially formed alone. The selective irradiation of sections of the gel through a mask leads to the formation of patterned multicomponent networks, in which either one or two networks can be present at a particular position with a high degree of spatial control.

  13. Single nucleotide polymorphisms of APOA1 gene and their relationship with serum apolipoprotein A-I concentrations in the native population of Assam

    PubMed Central

    Bora, Kaustubh; Pathak, Mauchumi Saikia; Borah, Probodh; Hussain, Md. Iftikar; Das, Dulmoni

    2015-01-01

    Background There is a growing interest in the role of allelic variants of the APOA1 gene in relation to a number of disorders. We described two common polymorphisms of the APOA1 gene, G-75A and C+83T and investigated their potential influence on the serum apolipoprotein A-I (apo A-I) levels in the native population of Assam — a region that is ethnically distinct and from where no information is hitherto available. Methods Blood samples were collected from 150 healthy volunteers. Apo A-I levels were estimated by immunoturbidometry. Genotyping was done by a PCR-RFLP method that involved DNA extraction from whole blood, followed by polymerase chain reaction and digestion of the PCR product by MspI restriction enzyme, and analysis of fragment sizes in 12% polyacrylamide gel. Results The GG variant at G-75A locus and CC variant at C+83T locus were the most prevalent. GG/CC was the most common combination. Homozygous TT genotype was not detected in any of the subjects. The rare allele frequencies for the G-75A and C+83T sites were found to be 0.22 and 0.06 respectively, which significantly differed from those reported in some other populations in neighbouring regions. Serum apo A-I concentrations did not vary significantly across the detected genotypes. These findings were consistent in both sexes. Conclusion We described the distribution of the G-75A and C+83T polymorphisms of the APOA1 gene in the population of Assam for the first time. These polymorphisms were not found to directly influence apo A-I concentrations in this population either individually or synergistically. PMID:26702398

  14. Synthesis of a novel affinity gel for the purification of carbonic anhydrases.

    PubMed

    Bozdag, Murat; Isik, Semra; Beyaztas, Serap; Arslan, Oktay; Supuran, Claudiu T

    2015-04-01

    A new affinity gel was synthesized for the purification of carbonic anhydrase (CA, EC 4.2.1.1) isozymes from erythrocytes. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on ethylenediamine was covalently attached via CNBr activation, followed by reaction with the CA inhibitor 4-isothiocyanato-benzenesulfonamide. The derivatized gel incorporated thioureido-benzenesulfonamide moieties as CA ligand. The binding capacity of the new affinity gel was determined at different temperatures, pH values, ionic strengths and elution buffers. The maximum binding of various CAs was achieved at 25 °C with pH 8.5 and ionic strength around 0.4. The overall purifications for human (h) hCA I and hCA II were 672- and 580-fold, and with 62 and 43% yields, respectively. SDS-polyacrylamide gel electrophoresis showed single bands for each purified isozymes, corresponding to a molecular weight of approx. 29 kDa. This is an easily obtainable, efficient and robust affinity gel, useful for the purification of many other α-CAs. PMID:24939102

  15. Monte Carlo modeling of a conventional X-ray computed tomography scanner for gel dosimetry purposes.

    PubMed

    Hayati, Homa; Mesbahi, Asghar; Nazarpoor, Mahmood

    2016-01-01

    Our purpose in the current study was to model an X-ray CT scanner with the Monte Carlo (MC) method for gel dosimetry. In this study, a conventional CT scanner with one array detector was modeled with use of the MCNPX MC code. The MC calculated photon fluence in detector arrays was used for image reconstruction of a simple water phantom as well as polyacrylamide polymer gel (PAG) used for radiation therapy. Image reconstruction was performed with the filtered back-projection method with a Hann filter and the Spline interpolation method. Using MC results, we obtained the dose-response curve for images of irradiated gel at different absorbed doses. A spatial resolution of about 2 mm was found for our simulated MC model. The MC-based CT images of the PAG gel showed a reliable increase in the CT number with increasing absorbed dose for the studied gel. Also, our results showed that the current MC model of a CT scanner can be used for further studies on the parameters that influence the usability and reliability of results, such as the photon energy spectra and exposure techniques in X-ray CT gel dosimetry. PMID:26205316

  16. Label-free DNA hybridization detection with molecular beacon immobilized in photopolymerized acrylamide gel microarray

    NASA Astrophysics Data System (ADS)

    Wang, Hong; Li, Jiong; Liu, Quanjun; Liu, Heping; Lu, Zuhong; Zhu, Jijun

    2001-10-01

    Photopolymerized acrylamide gel microarray used to immobilize the molecular beacon for label free DNA hybridization detection is described in this paper. Polyacrylamide gel microarray was prepared by UV photopolymerization of 4% acrylamide in 40% glycerol, 0.002%methylene blue, 0.012% TEMED and 0.1M phosphate buffer (PHequals7) with a Relpel-silane pretreated quartz mask. This kind of three-dimensional gel microarray provides more than 100 times great immobilization capacity. The hybridization and other processes with it resemble a homogeneous liquid phase reaction rather than a heterogeneous liquid-solid interface reaction. The specially designed molecular beacons contain a 15 base loop sequence with 5 base pair stem, a 20 base thymine as spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the 20 base thymine is used to minimize destability caused by 5'-end immobilization. Confocal microscope was used to investigate the fluorescence intensity of gel immobilized molecular beacon probes. After hybridization we can easily distinguish complementary and noncomplementay targets with gel-immobilized molecular beacon probes. Image analysis showed that the fluorescence intensity ratio of complementary to noncomplementay probes is great than 5. The potential applications of gel-immobilized molecular beacon microarray are mutation detection, pathogenic detection, etc. in a parallel, cost saving and label-free detection way.

  17. Elastodynamics of gels

    SciTech Connect

    Johnson, D.L.

    1982-08-01

    The mechanical properties of gels are analyzed in terms of models which treat the three-dimensional displacements of solid and fluid parts separately and on an equal footing, with no assumptions regarding smallness of concentration or even weakness of the solid skeleton frame. Thus, a unified description of the normal modes in a gel is presented; parameters deduced from measurement on a given mode are shown to influence the properties of the others. It is still assumed, however, that the fluid and solid components can be described by their bulk densities, elastic moduli, etc. This picture is discussed in light of experimental results. 37 references.

  18. Characterization of Network Structure of Polyacrylamide Based Hydrogels Prepared By Radiation Induced Polymerization

    SciTech Connect

    Mahmudi, Naim; Sen, Murat; Gueven, Olgun; Rendevski, Stojan

    2007-04-23

    In this study network structure of polyacrylamide based hydrogels prepared by radiation induced polymerization has been investigated. Polyacrylamide based hydrogels in the rod form were prepared by copolymerization of acrylamide(AAm) with hydroxyl ethyl methacrylate(HEMA) and methyl acrylamide(MAAm) in the presence of cross-linking agent and water by gamma rays at ambient temperature. Molecular weight between cross-links and effective cross-link density of hydrogels were calculated from swelling as well as shear modulus data obtained from compression tests. The results have shown that simple compression analyses can be used for the determination of effective cross-link density of hydrogels without any need to some polymer-solvent based parameters as in the case of swelling based determinations. Diffusion of water into hydrogels was examined by analyzing water absorption kinetics and the effect of network, structure on the diffusion type and coefficient was discussed.

  19. EFFECT OF DEXTRAN-graft-POLYACRYLAMIDE INTERNAL STRUCTURE ON FLOCCULATION PROCESS PARAMETERS

    SciTech Connect

    Bezugla, T.; Kutsevol, N.; Shyichuk, A.; Ziolkowska, D.

    2008-08-28

    Dextran-graft-Polyacrylamide copolymers (D-g-PAA) of brush-like architecture were tested as flocculation aids in the model kaolin suspensions. Due to expanded conformation the D-g-PAA copolymers are more effective flocculants than individual PAA with close molecular mass. The internal structure of D-g-PAA copolymers which is determined by number and length of grafted PAA chains, the distance between grafts, etc., has the significant influence on flocculation behavior of such polymers.

  20. Effects of polyacrylamide soil conditioner on the iron status of soybean plants. [Glycine max

    SciTech Connect

    Wallace, A.; Wallace, G.A.; Abouzamzam, A.M.; Char, J.W.

    1986-05-01

    An iron-inefficient cultivar of soybean (Glycine max L. Merr. Bragg cv. PI-54619-5-1 was grown in two different calcareous soils, a Natrargid and a Torrifluvents, to determine if improvement of soil aeration with a synthetic polyacrylamide as a soil conditioner would decrease the tendency of the cultivar to lime-induced chlorosis. The results suggest that when soil is well aerated with good drainage from use of the soil conditioner, the iron status of plants is improved.

  1. A relational Fuzzy C-Means algorithm for detecting protein spots in two-dimensional gel images.

    PubMed

    Rashwan, Shaheera; Faheem, Talaat; Sarhan, Amany; Youssef, Bayumy A B

    2010-01-01

    Two-dimensional polyacrylamide gel electrophoresis of proteins is a robust and reproducible technique. It is the most widely used separation tool in proteomics. Current efforts in the field are directed at the development of tools for expanding the range of proteins accessible with two-dimensional gels. Proteomics was built around the two-dimensional gel. The idea that multiple proteins can be analyzed in parallel grew from two-dimensional gel maps. Proteomics researchers needed to identify interested protein spots by examining the gel. This is time consuming, labor extensive and error prone. It is desired that the computer can analyze the proteins automatically by first detecting, then quantifying the protein spots in the 2D gel images. This paper focuses on the protein spot detection and segmentation of 2D gel electrophoresis images. We present a new technique for segmentation of 2D gel images using the Fuzzy C-Means (FCM) algorithm and matching spots using the notion of fuzzy relations. Through the experimental results, the new algorithm was found out to detect protein spots more accurately, then the current known algorithms. PMID:20865504

  2. Native Education 101: Basic Facts about American Indian, Alaska Native, and Native Hawaiian Education

    ERIC Educational Resources Information Center

    National Indian Education Association, 2008

    2008-01-01

    This document contains information about education of indigenous American peoples, including: (1) Demographics; (2) Reservations and Native Lands Map; (3) Important Community Issues and Concepts; (4) Educational Issues for Native Students; (5) Type of Schools for Native Students; (6) Indian Education Legislation and Executive Orders; (7)

  3. Staining of proteins after isoelectric focusing in gels by new procedures.

    PubMed

    Vesterberg, O; Hansén, L; Sjösten, A

    1977-03-28

    Four procedures are described for staining of protein bands after isoelectric focusing in polyacrylamide gels. In one procedure, Coomassie Brilliant Blue R 250 is used whereas in the others the G 250 form is used in an aqueous solution of perchloric acid. By using isoelectric focusing and the new staining procedures it is possible to detect less than 0.2 microng of many proteins. One of the procedures is especially rapid, allowing detection of proteins without destaining. Procedures for preservation of the protein patterns and determination of proteins by scanning in a densitometer are also described. PMID:849455

  4. Laboratory and Field Evaluations of Polyacrylamide Hydrogel Baits Against Argentine Ants (Hymenoptera: Formicidae).

    PubMed

    Rust, Michael K; Soeprono, Andrew; Wright, Sarajean; Greenberg, Les; Choe, Dong-Hwan; Boser, Christina L; Cory, Coleen; Hanna, Cause

    2015-06-01

    The development of effective baits to control the Argentine ant, Linepithema humile (Mayr), has been problematic because foragers prefer sweet liquids, while many toxicants are insoluble in water and liquid baits are generally difficult to deliver. The incorporation of thiamethoxam and sucrose solutions into a water-absorbing polyacrylamide hydrogel provides a unique and novel carrier and method of application for liquid baits. Formulations of thiamethoxam affected the size of the hydrogels, and sucrose solutions containing 0.0003% technical thiamethoxam provided hydrogels as large as those made with 25% sucrose solution or deionized water. Concentrations of thiamethoxam as low as 0.000075% in the hydrogels provided 50% kill of workers within 3 d in a laboratory setting. In small colony studies, baiting with 0.00015 and 0.000075% thiamethoxam hydrogels provided 100% mortality of workers and queens within 8 d. An enzyme-linked immunosorbent assay indicated that thiamethoxam was absorbed into the interior of the polyacrylamide matrix. The water loss rates of the hydrogels were dependent upon the relative humidity. Polyacrylamide hydrogels with >50% water loss were less attractive to ants. Field studies in highly infested areas indicated that concentrations of 0.0006 or 0.0018% thiamethoxam were more effective than 0.00015%. Hydrogels may provide a cost-effective alternative to providing aqueous baits to control Argentine ants. PMID:26470250

  5. Isolation and sequence analysis of proteins from mouse forebrain using two-dimensional gel electrophoresis coupled to high-pressure liquid extrusion.

    PubMed

    DeWald, D B; Pearson, J D

    1989-08-01

    This report presents a technique for recovery of mouse forebrain proteins from two-dimensional sodium dodecyl sulfate-polyacrylamide gels for subsequent primary structure determination. Proteins were visualized by Coomassie staining or salt precipitation and manually cut out of the gel. Excised spots were minced and loaded into an empty precolumn of a reversed-phase high-performance liquid chromatography system. Purified protein was extruded from a gel matrix by pressurized liquid, then separated from gel contaminants by reversed-phase gradient elution, and finally collected in siliconized tubes or on polybrene-coated filter disks for gas-phase sequencing. Several mouse and rat forebrain proteins were purified by this method and sequenced. Three previously unidentified mouse brain proteins with molecular weights of 4,000, 12,000, and 18,500 were partially sequenced and three hemoglobin fragments were structurally identified and mapped. Ribonuclease A, myoglobin, adrenocorticotropin, and bovine somatotropin were also subjected to two-dimensional (2-D) analysis and partially sequenced. Recovery values of 27-95% were obtained for extruded 14C-labeled ribonuclease, carbonic anhydrase, and bovine serum albumin out of sodium dodecyl sulfate-polyacrylamide gel electrophoretic gels. Losses resulting from the multiple handling steps of a 2-D gel separation process were also investigated. Recoveries of 12-17%, as determined by sequencing signals, were achieved. These latter recovery values reflect overall losses incurred in gel-focusing, gel-sizing, staining, destaining, high-pressure liquid extrusion, and N-terminal blockage. This work demonstrates that an array of protein spots can be systematically identified or defined by partial sequencing after high-pressure liquid extrusion from a 2-D gel matrix.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2817364

  6. Neuropsychological performance of native versus non-native English speakers.

    PubMed

    Kisser, Jason E; Wendell, Carrington R; Spencer, Robert J; Waldstein, Shari R

    2012-11-01

    Relatively little is known about differences in English-administered, clinical neuropsychological test performance between native versus non-native English speakers, with prior literature yielding mixed findings. The purpose of this study was to examine the performance of native and non-native English speakers with similar age and educational backgrounds on a variety of cognitive tests. Participants were 153 university students (115 native and 38 non-native English speakers) who completed a neuropsychological battery during two testing sessions. Multiple regression analyses examined relations of native language to cognitive performance after adjustment for age, education, sex, and depressive symptomatology. Results showed that native English speakers outperformed non-native English speakers on several language-mediated tasks-Letter and Category Fluency and the Cognitive Estimation Test-as well as Trails A (p's < .05). The two groups performed similarly on tests of executive functions, perceptuo-motor speed, verbal memory, and visuospatial abilities. These results suggest that non-native English language may have a negative influence predominantly on language-dependent tasks. PMID:22985952

  7. Rheology of Active Gels

    NASA Astrophysics Data System (ADS)

    Chen, Daniel

    2015-03-01

    Active networks drive a diverse range of critical processes ranging from motility to division in living cells, yet a full picture of their rheological capabilities in non-cellular contexts is still emerging, e.g., How does the rheological response of a network capable of remodeling under internally-generated stresses differ from that of a passive biopolymer network? In order to address this and other basic questions, we have engineered an active gel composed of microtubules, bidirectional kinesin motors, and molecular depletant that self-organizes into a highly dynamic network of active bundles. The network continually remodels itself under ATP-tunable cycles of extension, buckling, fracturing, and self-healing. Using confocal rheometry we have simultaneously characterized the network's linear and non-linear rheological responses to shear deformation along with its dynamic morphology. We find several surprising and unique material properties for these active gels; most notably, rheological cloaking, the ability of the active gel to drive large-scale fluid mixing over several orders of flow magnitude while maintaining an invariant, solid-like rheological profile and spontaneous flow under confinement, the ability to exert micro-Newton forces to drive persistent directed motion of the rheometer tool. Taken together, these results and others to be discussed highlight the rich stress-structure-dynamics relationships in this class of biologically-derived active gels.

  8. Enzymatically active biomimetic micropropellers for the penetration of mucin gels

    PubMed Central

    Walker, Debora; Käsdorf, Benjamin T.; Jeong, Hyeon-Ho; Lieleg, Oliver; Fischer, Peer

    2015-01-01

    In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacterium Helicobacter pylori has developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus. PMID:26824056

  9. Transdermal delivery of paeonol using cubic gel and microemulsion gel

    PubMed Central

    Luo, Maofu; Shen, Qi; Chen, Jinjin

    2011-01-01

    Background The aim of this study was to develop new systems for transdermal delivery of paeonol, in particular microemulsion gel and cubic gel formulations. Methods Various microemulsion vehicles were prepared using isopropyl myristate as an oil phase, polyoxyethylated castor oil (Cremophor® EL) as a surfactant, and polyethylene glycol 400 as a cosurfactant. In the optimum microemulsion gel formulation, carbomer 940 was selected as the gel matrix, and consisted of 1% paeonol, 4% isopropyl myristate, 28% Cremophor EL/polyethylene glycol 400 (1:1), and 67% water. The cubic gel was prepared containing 3% paeonol, 30% water, and 67% glyceryl monooleate. Results A skin permeability test using excised rat skins indicated that both the cubic gel and microemulsion gel formulations had higher permeability than did the paeonol solution. An in vivo pharmacokinetic study done in rats showed that the relative bioavailability of the cubic gel and microemulsion gel was enhanced by about 1.51-fold and 1.28-fold, respectively, compared with orally administered paeonol suspension. Conclusion Both the cubic gel and microemulsion gel formulations are promising delivery systems to enhance the skin permeability of paeonol, in particular the cubic gel. PMID:21904450

  10. Frequency of Occurrence of Native Hapten Among Enterobacterial Species

    PubMed Central

    Anacker, R. L.; Bickel, W. D.; Haskins, W. T.; Milner, K. C.; Ribi, E.; Rudbach, J. A.

    1966-01-01

    Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), W. D. Bickel, W. T. Haskins, K. C. Milner, E. Ribi, and J. A. Rudbach. Frequency of occurrence of native hapten among enterobacterial species. J. Bacteriol. 91:1427–1433. 1966.—Smooth cultures of representative Enterobacteriaceae were screened for the presence of native hapten, a substance previously extracted with trichloroacetic acid from the protoplasmic fraction of one strain each of Escherichia coli O111:B4 and O113. Trichloroacetic acid extracts of protoplasmic fractions of the cells were analyzed for chemical composition, for constituent sugars by paper chromatography, for immunochemical relationship to endotoxin purified by gel filtration, for sedimentation behavior, and for pyrogenicity in rabbits and lethal toxicity in chick embryos. Extracts from two of three additional strains of E. coli O113, all five additional strains of E. coli O111:B4, and one strain each of E. coli O26:B6 and O55:B5 were similar to previously described native hapten in chemical composition, sedimentation properties (S20,w, 3.7 to 5.2), biological potency (usually less than 0.1% that of corresponding endotoxin), and immunochemical relationship to endotoxin. Extracts of one strain each of E. coli O127:B8, Serratia marcescens, Citrobacter freundii, Salmonella enteritidis, and of two lipopolysaccharide-deficient mutants of S. enteritidis differed from typical native hapten. The biosynthetic relationship of native hapten to endotoxin has not yet been revealed. Images PMID:5326701

  11. Physicochemical modification of native and extruded wheat flours by enzymatic amylolysis.

    PubMed

    Martínez, Mario M; Pico, Joana; Gómez, Manuel

    2015-01-15

    Enzymatic hydrolysis could be an alternative way to modify flour functionality. The effect of two different enzymes, α-amylase and amyloglucosidase, and their combination on microstructure, oligosaccharide content, crystalline order, pasting, gel hydration, and colour properties of native and extruded wheat flours was investigated. Micrographs showed different mechanisms of actuation of the different enzymes on native and extruded flours, achieving greater than 300% and 500% increases of glucose and maltose contents, respectively, in extruded flours compared with their native counterparts. Native flours displayed higher values of water absorption capacity and swelling power than extruded flours. Flours treated by a combination of amylase and amyloglucosidase showed low swelling power. Regarding colour, native flours were darker and more reddish than extruded flours, whereas flours treated by amyloglucosidase, and therefore had a higher glucose content, were darker and more reddish. PMID:25149010

  12. Linear electrochemical gel actuators

    NASA Astrophysics Data System (ADS)

    Goswami, Shailesh; McAdam, C. John; Hanton, Lyall R.; Moratti, Stephen C.

    2011-11-01

    By using electroactive monomers it is possible to produce gels that respond to oxidation or reduction by swelling and deswelling in the presence of solvent. By the inclusion of an appropriate biasing element such as a spring, it is possible to produce linear, reversible actuation. The process can be driven electrochemically in a standard cell, with driving voltages under +/- 1 V. For many systems, the intrinsic conductivity of the gel, leading to poor or no performance. This can be overcome by blending conductive carbon nanotubes at 1% concentration, which give reasonable conductivity without affecting mechanical performance. Extensions of up to 40% are possible, against an external pressure of 30 kPa. The process is slow, taking up to 160 minutes per cycle due to slow ionic diffusion. The electrochemical cell can be cycled many times without degradation.

  13. Linear electrochemical gel actuators

    NASA Astrophysics Data System (ADS)

    Goswami, Shailesh; McAdam, C. John; Hanton, Lyall R.; Moratti, Stephen C.

    2012-04-01

    By using electroactive monomers it is possible to produce gels that respond to oxidation or reduction by swelling and deswelling in the presence of solvent. By the inclusion of an appropriate biasing element such as a spring, it is possible to produce linear, reversible actuation. The process can be driven electrochemically in a standard cell, with driving voltages under +/- 1 V. For many systems, the intrinsic conductivity of the gel, leading to poor or no performance. This can be overcome by blending conductive carbon nanotubes at 1% concentration, which give reasonable conductivity without affecting mechanical performance. Extensions of up to 40% are possible, against an external pressure of 30 kPa. The process is slow, taking up to 160 minutes per cycle due to slow ionic diffusion. The electrochemical cell can be cycled many times without degradation.

  14. Introduced brown trout alter native acanthocephalan infections in native fish.

    PubMed

    Paterson, Rachel A; Townsend, Colin R; Poulin, Robert; Tompkins, Daniel M

    2011-09-01

    1. Native parasite acquisition provides introduced species with the potential to modify native host-parasite dynamics by acting as parasite reservoirs (with the 'spillback' of infection increasing the parasite burdens of native hosts) or sinks (with the 'dilution' of infection decreasing the parasite burdens of native hosts) of infection. 2. In New Zealand, negative correlations between the presence of introduced brown trout (Salmo trutta) and native parasite burdens of the native roundhead galaxias (Galaxias anomalus) have been observed, suggesting that parasite dilution is occurring. 3. We used a multiple-scale approach combining field observations, experimental infections and dynamic population modelling to investigate whether native Acanthocephalus galaxii acquisition by brown trout alters host-parasite dynamics in native roundhead galaxias. 4. Field observations demonstrated higher infection intensity in introduced trout than in native galaxias, but only small, immature A. galaxii were present in trout. Experimental infections also demonstrated that A. galaxii does not mature in trout, although parasite establishment and initial growth were similar in the two hosts. Taken together, these results support the hypothesis that trout may serve as an infection sink for the native parasite. 5. However, dynamic population modelling predicts that A. galaxii infections in native galaxias should at most only be slightly reduced by dilution in the presence of trout. Rather, model exploration indicates parasite densities in galaxias are highly sensitive to galaxias predation on infected amphipods, and to relative abundances of galaxias and trout. Hence, trout presence may instead reduce parasite burdens in galaxias by either reducing galaxias density or by altering galaxias foraging behaviour. PMID:21426342

  15. Two-dimensional Gel Electrophoresis (2DE)

    NASA Astrophysics Data System (ADS)

    Kłodzińska, Ewa; Buszewski, Bogusław

    The chemical compounds, which are present in the environment, increasingly cause bad effects on health. The most serious effects are tumors and various mutations at the cellular level. Such compounds, from the analytical point of view, can serve the function of biomarkers, constituting measurable changes in the organism's cells and biochemical processes occurring therein. The challenge of the twenty-first century is therefore searching for effective and reliable methods of identification of biomarkers as well as understanding bodily functions, which occur in living organisms at the molecular level. The irreplaceable tool for these examinations is proteomics, which includes both quality and quantity analysis of proteins composition, and also makes it possible to learn their functions and expressions. The success of proteomics examinations lies in the usage of innovative analytical techniques, such as electromigration technique, two-dimensional electrophoresis in polyacrylamide gel (2D PAGE), liquid chromatography, together with high resolution mass spectrometry and bio-informatical data analysis. Proteomics joins together a number of techniques used for analysis of hundreds or thousands of proteins. Its main task is not the examination of proteins inside the particular tissue but searching for the differences in the proteins' profile between bad and healthy tissues. These differences can tell us a lot regarding the cause of the sickness as well as its consequences. For instance, using the proteomics analysis it is possible to find relatively fast new biomarkers of tumor diseases, which in the future will be used for both screening and foreseeing the course of illness. In this chapter we focus on two-dimensional electrophoresis because as it seems, it may be of enormous importance when searching for biomarkers of cancer diseases.

  16. [Agarose gel isoelectric focusing: application to the study of abnormalities of immunoglobulin clonality in CSF and serum].

    PubMed

    Lebrun-Fourcy, C; Rondot, J; Revol, C; Renversez, J C

    1996-01-01

    Since it is quite difficult to commonly use isoelectric focusing (IEF) of proteins in polyacrylamide gel for biological diagnosis, we have developed a method based on IEF in agarose gel, to split proteins from sera and cerebrospinal fluid (CSF). A prefocalisation at low voltage (250 V) is made on a custom thin gel of agarose (0.5 mm) containing some carrier ampholytes (pH 5-9). After deposition of biological samples, the gel is run at 500 V, thereafter at 1200 V. After focusing, the gel is fixed before being coloured by a simplified silver staining technique. In order to demonstrate the good resolution of the immunoglobulines (Ig) in the pH gradient, a transfer on a nitrocellulose membrane followed by an immunofixation was carried out from unstained gels after IEF. This separation on agarose gel shows several advantages, ie its speed (3H total), its lack of toxicity, its sensibility and its reproductibility. It is specially well suited for the diagnosis of diseases characterised by oligoclonal or monoclonal Ig, particularly those found in the CSF during neurologic diseases like multiple sclerosis. Several examples of focused sera and CSF are reviewed in the paper. PMID:8952725

  17. An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection.

    PubMed

    Chen, Xiaojia; Ugaz, Victor M

    2006-02-01

    In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings. PMID:16342324

  18. Development of a gel formulation of formic acid for control of parasitic mites of honey bees.

    PubMed

    Kochansky, J; Shimanuki, H

    1999-09-01

    Formic acid has been used in various countries for the control of parasitic mites of honey bees (Apis mellifera), particularly the Varroa mite (Varroa jacobsoni) and the tracheal mite (Acarapis woodi). Its corrosivity and consequent fear of liability have precluded commercial interest in the United States, and its rapid vaporization requires frequent reapplication. We have developed a gel formulation of formic acid which provides controlled release over 2-3 weeks and improves the convenience and safety of handling of formic acid. The strong acidity of formic acid restricts the choice of gelling agents; vegetable gellants such as agar are destroyed, and bentonite clay derivatives do not gel, even with high-shear mixing. Polyacrylamides lead to viscous liquids lacking thixotropic properties. High-molecular-weight poly(acrylic acids) and fumed silicas provided gels with suitable physical characteristics. The poly(acrylic acid) gels were difficult to mix and gave slower and nonlinear release behavior, while the fumed silica gels were easy to prepare and linear in formic acid vaporization. PMID:10552733

  19. The detection of oligonucleotide N3'-->P5' phosphoramidate/RNA duplexes with capillary gel electrophoresis.

    PubMed

    DeDionisio, L; Raible, A M; Gryaznov, S M

    1998-06-01

    Oligonucleotide N3'-->P5' phosphoramidates (3'-phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single-stranded RNA fragments utilizing 3'-phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3'-Phosphoramidate 9-mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3'-phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3'-phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation. PMID:9694262

  20. Verification of motion induced thread effect during tomotherapy using gel dosimetry

    NASA Astrophysics Data System (ADS)

    Edvardsson, Anneli; Ljusberg, Anna; Ceberg, Crister; Medin, Joakim; Ambolt, Lee; Nordström, Fredrik; Ceberg, Sofie

    2015-01-01

    The purpose of the study was to evaluate how breathing motion during tomotherapy (Accuray, CA, USA) treatment affects the absorbed dose distribution. The experiments were carried out using gel dosimetry and a motion device simulating respiratory-like motion (HexaMotion, ScandiDos, Uppsala, Sweden). Normoxic polyacrylamide gels (nPAG) were irradiated, both during respiratory-like motion and in a static mode. To be able to investigate interplay effects the static absorbed dose distribution was convolved with the motion function and differences between the dynamic and convolved static absorbed dose distributions were interpreted as interplay effects. The expected dose blurring was present and the interplay effects formed a spiral pattern in the lower dose volume. This was expected since the motion induced affects the preset pitch and the theoretically predicted thread effect may emerge. In this study, the motion induced thread effect was experimentally verified for the first time.

  1. Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

    PubMed

    Bruin, G J; Börnsen, K O; Hüsken, D; Gassmann, E; Widmer, H M; Paulus, A

    1995-08-11

    The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented. PMID:7581844

  2. Wetting mechanisms of gel-based controlled-release fertilizers.

    PubMed

    Shavit, U; Reiss, M; Shaviv, A

    2003-02-14

    The release mechanism of gel-based controlled release fertilizers (CRFs) involves water penetration into dry mixtures of fertilizers and gel forming polymers. Water penetration provides an upper limit to the whole release process. Where wetting prediction is often based on models that describe the flow of the liquid phase, vapor motion may become significant when a sharp wetting front exists. In this study we examine the role of vapor and fluid flows in the wetting process of CRFs consisting of urea or KNO(3) mixed with polyacrylamide (PAM). Vapor adsorption isotherms were obtained for typical fertilizer-PAM mixtures. Wetting and release experiments were conducted by dividing the CRFs into regions alternately filled with a pure fertilizer and mixtures of PAM and fertilizer. The experiments were designed in such a way that when the wetting front reaches a mixtures interface, its motion depends on the gradient imposed by the difference in osmotic potential (OP). The coupled equations of vapor and liquid flow in initially dry conditions were solved numerically to demonstrate the conceptual understanding gained by the experiments. The results show that wetting front motion is affected by transport and adsorption of vapor. It was also shown that the release rate is different when wetting is governed by vapor flow or by liquid flow. The release pattern from a multi-regions device was consistent with the wetting pattern, demonstrating the possibility to tailor the release according to periods of peak demand. PMID:12586505

  3. Polyacrylamide-hydroxyapatite composite: Preparation, characterization and adsorptive features for uranium and thorium

    SciTech Connect

    Baybas, Demet; Ulusoy, Ulvi

    2012-10-15

    The composite of synthetically produced hydroxyapatite (HAP) and polyacrylamide was prepared (PAAm-HAP) and characterized by BET, FT-IR, TGA, XRD, SEM and PZC analysis. The adsorptive features of HAP and PAAm-HAP were compared for UO{sub 2}{sup 2+} and Th{sup 4+}. The entrapment of HAP into PAAm-HAP did not change the structure of HAP. Both structures had high affinity to the studied ions. The adsorption capacity of PAAm-HAP was than that of HAP. The adsorption dependence on pH and ionic intensity provided supportive evidences for the effect of complex formation on adsorption process. The adsorption kinetics was well compatible to pseudo second order model. The values of enthalpy and entropy changes were positive. Th{sup 4+} adsorption from the leachate obtained from a regional fluorite rock confirmed the selectivity of PAAm-HAP for this ion. In consequence, PAAm-HAP should be considered amongst favorite adsorbents for especially deposition of nuclear waste containing U and Th, and radionuclide at secular equilibrium with these elements. - Graphical abstract: SEM images of hydroxyapatite (HAP) and polyacrylamide-hydroxyapatite (PAAm-HAP), and the adsorption isotherms for Uranium and Thorium. Highlights: Black-Right-Pointing-Pointer Composite of PAAm-HAP was synthesized from hydroxyapatite and polyacrylamide. Black-Right-Pointing-Pointer The materials were characterized by BET, FT-IR, XRD, SEM, TGA and PZC analysis. Black-Right-Pointing-Pointer HAP and PAAm-HAP had high sorption capacity and very rapid uptake for UO{sub 2}{sup 2+} and Th{sup 4+}. Black-Right-Pointing-Pointer Super porous PAAm was obtained from PAAm-HAP after its removal of HAP content. Black-Right-Pointing-Pointer The composite is potential for deposition of U, Th and its associate radionuclides.

  4. Ceric ion initiated synthesis of polyacrylamide grafted oatmeal: Its application as flocculant for wastewater treatment.

    PubMed

    Bharti, Srijita; Mishra, Sumit; Sen, Gautam

    2013-04-01

    Polyacrylamide grafted oatmeal (OAT-g-PAM) was synthesized by conventional method. The grafting of the PAM chains on the biomaterial backbone was confirmed through intrinsic viscosity study, FTIR spectroscopy, elemental analysis (C, H, N, S and O), SEM morphology and TGA study. The intrinsic viscosity of oatmeal appreciably improved on grafting of PAM chains, thus resulting grafted product with potential application as superior viscosifier. Further, flocculation efficacy of the graft copolymer was studied in coal fine suspension, kaolin suspension, iron-ore suspension and then in municipal wastewater through 'jar test' procedure. PMID:23499093

  5. A novel polymeric flocculant based on polyacrylamide grafted inulin: aqueous microwave assisted synthesis.

    PubMed

    Rahul, Rahul; Jha, Usha; Sen, Gautam; Mishra, Sumit

    2014-01-01

    Polyacrylamide grafted inulin (In-g-PAM) was synthesized via aqueous microwave assisted method (using ceric ammonium nitrate in synergism with microwave in aqueous medium). The intended grafting of the PAM chains on polysaccharide backbone was confirmed through standard physicochemical characterization techniques, namely intrinsic viscosity measurement, Fourier transform infrared (FTIR) spectroscopy, elemental analysis (C, H, N and O), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) studies. Flocculation efficacy of various grades of synthesized grafted product was studied in coal fines suspension, in relation to inulin (parent polysaccharide). This was done utilizing jar test and settling test procedure, towards possible application as a flocculant for coal washery effluents. PMID:24274474

  6. Instabilities with polyacrylamide solution in small and large aspect ratios Taylor-Couette systems

    NASA Astrophysics Data System (ADS)

    Smieszek, M.; Crumeyrolle, O.; Mutabazi, I.; Egbers, C.

    2008-11-01

    We have investigated the stability of viscoelastic polyacrylamide solution in Taylor-Couette system with different aspect ratios. The first instability modes observed in a Taylor-Couette system with Γ = 10 were TVF and WVF, as for Newtonian fluid. At higher Taylor numbers moving vortices occur, a wavy mode with non-stationary vortex size. In the Taylor-Couette system with Γ = 45.9 we note a coexistence of various instability modes. In addition to TVF, counterpropagating waves developed at the transition from the base state flow. At higher Taylor number values Taylor vortices of different sizes occurred. Reduced amplitude Wavy vortex flow has also been observed.

  7. Effects of polyacrylamide, biopolymer, and biochar on decomposition of soil organic matter and 14C-labeled plant residues as determined by enzyme activities

    NASA Astrophysics Data System (ADS)

    Mahmoud Awad, Yasser; Ok, Young Sik; Kuzyakov, Yakov

    2014-05-01

    Application of polymers for the improvement of aggregate structure and reduction of soil erosion may alter the availability and decomposition of plant residues. In this study, we assessed the effects of anionic polyacrylamide (PAM), synthesized biopolymer (BP), and biochar (BC) on the decomposition of 14C-labeled maize residue in sandy and sandy loam soils. Specifically, PAM and BP with or without 14C-labeled plant residue were applied at 400 kg ha-1, whereas BC was applied at 5000 kg ha-1, after which the soils were incubated for 80 days at 22 oC. Initially, plant residue decomposition was much higher in untreated sandy loam soil than in sandy soil. Nevertheless, the stimulating effects of BP and BC on the decomposition of plant residue were more pronounced in sandy soil, where it accounted for 13.4% and 23.4% of 14C input, respectively, whereas in sandy loam soil, the acceleration of plant residue decomposition by BP and BC did not exceed 2.6% and 14.1%, respectively, compared to untreated soil with plant residue. The stimulating effects of BP and BC on the decomposition of plant residue were confirmed based on activities of β-cellobiohydrolase, β-glucosidase, and chitinase in both soils. In contrast to BC and BP, PAM did not increase the decomposition of native or added C in both soils.

  8. Association Study between BDNF Gene Polymorphisms and Autism by Three-Dimensional Gel-Based Microarray

    PubMed Central

    Cheng, Lu; Ge, Qinyu; Xiao, Pengfeng; Sun, Beili; Ke, Xiaoyan; Bai, Yunfei; Lu, Zuhong

    2009-01-01

    Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined with dual-color hybridization for association study of four BDNF polymorphisms with autism. All the SNPs in both patients and controls could be analyzed quickly and correctly. Among four SNPs, only C270T polymorphism showed significant differences in the frequency of the allele (χ2 = 7.809, p = 0.005) and genotype (χ2 = 7.800, p = 0.020). In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (χ2 = 28.19, p = 3.44e-005). We suggest that BDNF has a possible role in the pathogenesis of autism. The study also show that the polyacrylamide gel-based microarray combined with dual-color hybridization is a rapid, simple and high-throughput method for SNPs genotyping, and can be used for association study of susceptible gene with disorders in large samples. PMID:19582215

  9. Ohiyesa's Path: Reclaiming Native Education

    ERIC Educational Resources Information Center

    James, Adrienne Brant; Renville, Tammy

    2012-01-01

    As Natives have assumed increasing authority and responsibility for tribal and federally funded and administered schools, a more balanced and enlightened view is emerging. Notable among these events is the recognition of the critical need to shift emphasis to the untapped heritage of more recently recognized and acknowledged Native American…

  10. Native Americans: The First Campers.

    ERIC Educational Resources Information Center

    Dunn, Bonnie; Frebertshauser, Denise

    2002-01-01

    Questions are presented to help camps determine if their usage of American Indian culture truly honors Native Americans. Camps that plan to use Indian lore should research the tribe's name, location, symbols, legends, and living habits. A 5-day program is presented for enhancing campers' understanding of Native peoples and their relationship to…

  11. Native American Foods and Cookery.

    ERIC Educational Resources Information Center

    Taylor, Tom; Potter, Eloise F.

    Native Americans had a well-developed agriculture long before the arrival of the Europeans. Three staples--corn, beans, and squash--were supplemented with other gathered plants or cultivated crops such as white potatoes, sweet potatoes, pumpkins, and peanuts. Native Americans had no cows, pigs, or domesticated chickens; they depended almost…

  12. Native Americans as Sports Mascots.

    ERIC Educational Resources Information Center

    Muir, Sharon Pray

    1999-01-01

    Discusses the controversy over whether sport teams should use Native American logos, mascots, or native symbols. Suggests that by implementing role-reversal techniques (putting the nonnative people in the same place) uncaring sports fans may recognize the disrespect involved; offers a hypothetical newspaper article that illustrates the…

  13. Native Language Literacy Screening Device.

    ERIC Educational Resources Information Center

    Hudson River Center for Program Development, Glenmont, NY.

    The purpose the Native Language Literacy Screening Device (NLLSD) is to give English for Speakers of Other Languages (ESOL) practitioners a sense of the native language literacy levels of learners coming into their programs. This is worth knowing because when learners have had limited schooling in their first language instructional strategies used…

  14. Alaska Native Personal Leadership Program.

    ERIC Educational Resources Information Center

    Curtis, Steven; Curtis, Jane; Halfon, Linda; Holt, Teresa; Knapp, Marilyn; Konshin, Luda; Schroeder, Tim; Navitsky, Mary Anne; Sunde, Elaine

    1998-01-01

    Describes the Alaska Native Leadership Program (ANLP), designed to recruit and retain Alaska Native college students. The year-long program includes an orientation, a two-semester class on self-exploration, skill-building, educational and career paths, and social activities. Student satisfaction with the program is indicated. (MMU)

  15. Coyote's Eyes: Native Cognition Styles.

    ERIC Educational Resources Information Center

    Tafoya, Terry

    1982-01-01

    Using a Native American parable, compares the Standard Average European (SAE) world view with the Standard Native American (SNA) world view and the effects they have on education. Points out possible areas of interethnic confusion as a result of these two world views in communication dealing with cognitive schemes. (LC)

  16. Coyote's Eyes: Native Cognition Styles.

    ERIC Educational Resources Information Center

    Tafoya, Terry

    The document compares and contrasts the Standard Average European (SAE) and the Standard Native American (SNA) viewpoints with regard to fostering cognitive development in children. One basic difference is demonstrated by relating a story and noting that, in terms of Native American cognitive development, no further teaching would be done. In…

  17. Alaska Natives In Higher Education.

    ERIC Educational Resources Information Center

    Kohout, Karen; Kleinfeld, Judith

    This study examines changes in the entrance and success rates of Native students in Alaska colleges from 1963 to 1972, a time when special college recruitment and assistance programs were being developed. Information is based on the college records of those Natives who entered college for the first time at the University of Alaska at Fairbanks…

  18. Native American Foods and Cookery.

    ERIC Educational Resources Information Center

    Taylor, Tom; Potter, Eloise F.

    Native Americans had a well-developed agriculture long before the arrival of the Europeans. Three staples--corn, beans, and squash--were supplemented with other gathered plants or cultivated crops such as white potatoes, sweet potatoes, pumpkins, and peanuts. Native Americans had no cows, pigs, or domesticated chickens; they depended almost

  19. Ohiyesa's Path: Reclaiming Native Education

    ERIC Educational Resources Information Center

    James, Adrienne Brant; Renville, Tammy

    2012-01-01

    As Natives have assumed increasing authority and responsibility for tribal and federally funded and administered schools, a more balanced and enlightened view is emerging. Notable among these events is the recognition of the critical need to shift emphasis to the untapped heritage of more recently recognized and acknowledged Native American

  20. Vanishing native American dog lineages

    PubMed Central

    2011-01-01

    Background Dogs were an important element in many native American cultures at the time Europeans arrived. Although previous ancient DNA studies revealed the existence of unique native American mitochondrial sequences, these have not been found in modern dogs, mainly purebred, studied so far. Results We identified many previously undescribed mitochondrial control region sequences in 400 dogs from rural and isolated areas as well as street dogs from across the Americas. However, sequences of native American origin proved to be exceedingly rare, and we estimate that the native population contributed only a minor fraction of the gene pool that constitutes the modern population. Conclusions The high number of previously unidentified haplotypes in our sample suggests that a lot of unsampled genetic variation exists in non-breed dogs. Our results also suggest that the arrival of European colonists to the Americas may have led to an extensive replacement of the native American dog population by the dogs of the invaders. PMID:21418639