Optimization of the conditions for serum protein two-dimensional gel electrophoresis in peripheral venous blood of human being
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Objective To optimize the conditions for serum protein two-dimensional gel electrophoresis(2-DE)in peripherally venous blood of human being.Methods After albumin,IgG and salt were removed from serum samples,immobilized pH gradient isoelectric focusing(IEF)and vertical sodium dodecyl sulfonate polyacrylamide gel electrophoresis(SDS-PAGE)were applied to separate proteins.The amount of protein sample,pH range of IPG,the concentration of gel,the volume of sample hydration fluid,the IEF process parameters and other conditions were improved.Results With the conditions of dissolving 50 μg to100 μg proteins to form 350 μl sample fluid hydration,adjusting IPG pH from 4 to 7 and using 12% SDS-PAGE gel,as well as optimizing electrophoresis current,time and other parameters,suitable conditions for 2-DE were established and some ideal two dimensional maps of proteins were acquired.Conclusion The optimizated conditions for serum protein 2-DE in human being are the foundation for further study on serum differential proteomics.Cite
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Objective:To establish a two-dimensional polyacrylamide gel electrophoresis(2-DE) technique for serum study.Methods: After elimination of serum albumin and IgG was achieved by filtering with proteoprep blue albumin depletion kit,proteins of interest were precipitated with acetone pre-cooling and subjected to adequate dissolution.Isoelectric focusing electrophoresis(IEF) was employed as the first dimension and sodium dodecyl sulfate-polyacrylamide gel elecrophoresis(SDS-PAGE) the second dimension.After silver nitrate staining,protein spots were analyzed.Results:A steady 2-DE technique procedure was established under opitimized conditions.Conclusion:The established 2-DE technique of human serum proteins can be effecting applied in serum proteomics of diseases.
Silver stain
Difference gel electrophoresis
Sodium dodecyl sulfate
Polyacrylamide
Silver nitrate
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Objective Syndrome of plasma proteins to establish two-dimensional gel electrophoresis and its optimization. Methods Normal human plasmas was used in this study. The samples were treated with three different ways: removing albumin and IgG globulin, boiling and no treating. The samples were then analyzed by two-dimensional gel electrophoresis with different pH gradients. After staining with argenti nitras, the gels were scanned and analyzed by PDQuest software. Results The electrophoregram of normal human plasmas was obtained by two-dimensional gel-electrophoresis. By silver nitrate staining,the proteins SDS hot plasma sample of the entire two-dimensional gel-electrophoresis staining deep, scattered clear. Conclusion Because of little-losing, simple and good repeatability,the method of two-dimensional gel- electrophoresis using heating-SDS whole plasma sample is an effective means of the syndrome plasma proteomics research.
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The effects of starch concentration on the resolution of serum components by gel electrophoresis were investigated. Gels were prepared with starch contents varying from 11.8 to 20.6% (w/v). Pooled serum samples from man, rabbit, guinea pig, rat, mouse, and chicken were separated by vertical and two-dimensional electrophoresis in the discontinuous Tris–citrate and borate system of buffers. The resolution of serum components at the various starch levels was compared with the results obtained with the standard concentration (11.8%) recommended for separation in human serum. A general increase in the number of bands staining for protein was observed in gels with starch concentrations between 13.0 and 16.0%, although species differences were observed in regard to optimum starch concentrations. The maximum number of serum proteins detected by one-dimensional electrophoresis were, for the various species: man, 27; rabbit, 15; guinea pig, 22; rat, 21; mouse, 19; and chicken, 12.
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Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.
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Sodium dodecyl sulfate
Serum Albumin
Proteome
Human serum albumin
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