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Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis 总被引:3,自引:0,他引:3
A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems. 相似文献
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Andreas U. Freiburghaus 《Molecular biotechnology》1994,2(3):281-293
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex
protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across
a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by
electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation
according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl
sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic
membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the
protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity,
antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there
is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation
will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method
for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing
technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer
at a low level. 相似文献
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Michael J. Daniels David B. Archer Michael A. Stephens Rodney Townsend Jane M. Longland Jennifer Best 《Current microbiology》1980,3(6):377-380
Pseudomonas oxalaticus utilized sodium acetate or fructose, in addition to sodium formate known to be assimilated via the reductive pentose phosphate
pathway. The generation time during growth on fructose (2 h, 10 min) was considerably shorter than observed for other pseudomonads,
which sequentially utilize a phosphoenolpyruvate-dependent phosphotransferase system and 1-phosphofructoninase during growth
on fructose. In contrast, extracts prepared from fructose-grownP. oxalaticus contaned enzyme activities indicative of the Entner-Doudoroff pathway, while 1-phosphofructokinase was not found. Our studies
indicate thatP. oxalaticus may be useful as a model organism for studies of CO2 fixation. 相似文献
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J Zwaan 《Analytical biochemistry》1967,21(2):155-168
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A system for express electrophoretic separation of proteins in a thin layer of polyacrylamide gel covalently attached to a glass base has been proposed. A technology of covalent attachment of gel to a glass base has been developed and a small-scale instrument has been constructed which allows to carry out the separation, staining and electrophoregram washing off for 60 min. The quality of the cleaved protein mixtures is not worse than the electrophoregrams obtained using the "Phase System T. M." instrument of "Pharmacia". 相似文献
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Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours. 相似文献
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Comparison of plant cytoplasmic ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis 总被引:2,自引:2,他引:0
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The electrophoretic mobilities of the cytoplasmic ribosomal proteins of several species of plants were compared using two-dimensional electrophoresis. The total number of proteins as well as the number of acidic and basic proteins in individual species varied markedly. Of the species examined, Triticum aestivum had the highest number of basic cytoplasmic ribosomal proteins and Hordeum vulgare had less than half as many. However, marked similarities were noted in the electrophoretic mobilities of many of the proteins, especially for wheat, rye, and barley and for peas and beans. There was a statistically significant positive correlation between the numbers of basic proteins in the species and their chromosome number. 相似文献
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A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor. 相似文献
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Peptide production from proteins separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis 总被引:5,自引:0,他引:5
The development of amino acid sequencers with subnanomolar sensitivities has increased the need for both selective and highly efficient methods for both protein and peptide isolation. In this paper, we describe a simple procedure that utilizes the high resolving capacity of polyacrylamide gel electrophoresis to isolate a single target polypeptide, which can subsequently be subjected to proteolytic digestion and sequencing. Polypeptides are visualized in polyacrylamide gels as dodecyl sulfate/protein complexes, which are passively diffused from gel slices. Free dodecyl sulfate eluted with the protein solution is removed by KCl precipitation, allowing protein digestion with small amounts of trypsin or other proteolytic enzymes. Following enzymatic digestion, the peptide solution is made 6 M guanidine-HCl to remove interfering contaminants and thereby improve resolution of the digest by reverse-phase high-performance liquid chromatography. The peptides generated by this method are suitable for amino acid sequencing with good overall yields, averaging 15-30% on a gas-phase sequenator. The method described is useful for obtaining multiple peptide sequences from a single polypeptide isolated from a complex protein mixture. 相似文献
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The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping. 相似文献
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Base analysis of ribopolynucleotides by tritium incorporation following analytical polyacrylamide gel electrophoresis 总被引:8,自引:0,他引:8
A procedure is described for tritium base composition analysis of major and modified constituents of 4S RNA following extraction of the RNA from analytical slab gels. Gel electrophoresis and isolation of RNA are shown not to lead to chemical alterations of RNA bases, degradation or preferential losses of RNA species. 相似文献
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