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Gel electrophoresis

About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.


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TL;DR: It is concluded that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifREEzes produced by fish and insects.
Abstract: Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.

198 citations

Journal ArticleDOI
TL;DR: Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.
Abstract: Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.

198 citations

Journal ArticleDOI
TL;DR: The bifidogenic effect of the EPS was confirmed by culturing on selective medium and the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

198 citations

Journal ArticleDOI
TL;DR: The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.
Abstract: The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.

198 citations

Journal ArticleDOI
TL;DR: Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase.
Abstract: Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s. Images

198 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202364
2022116
2021108
2020104
2019120
2018147