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1.
A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques. 相似文献
2.
Comparative Proteome Analysis of Human Temporal Cortex Lobes by Two-Dimensional Electrophoresis and Identification of Selected Common Proteins 总被引:2,自引:0,他引:2
Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins. 相似文献
3.
This paper describes a new method for solubilization and partial purification of a Na+-dependent phlorizin receptor from dog kidney proximal convoluted tubule. Selective solubilization is carried out with 0.1% Na+-deoxycholate followed by complete solubilization with 0.5% deoxycholate. The 100,000 X g supernatant of the deoxycholate extract is then subjected to a combination of chromatofocusing and gel exclusion chromatography. Purification is monitored by a new column assay which permits detection of the Na+-dependent high affinity phlorizin receptor in solubilized preparations. Na+-dependent phlorizin binding exhibits the same characteristics on the column assay as in intact brush border vesicles. Binding is temperature-dependent, inhibited by proteolytic agents, Na+-dependent, and inhibited by excess cold phlorizin and D-glucose but not L-glucose. Quantitation of specific binding at different stages of the isolation procedure indicates a final purification of approximately 80-140-fold compared to intact brush border membrane fragments. Enrichment of specific phlorizin binding is paralleled by enrichment of a 61-66-kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It is postulated that this polypeptide contains both the Na and the sugar specific binding site and represents a subunit of the intact Na+-dependent glucose transporter from dog kidney proximal tubule brush border membrane. 相似文献
4.
Isolation and characterization of a new pancreatic polypeptide hormone. 总被引:11,自引:0,他引:11
A method is described for isolation, from chicken pancreas, of an avian pancreatic polypeptide which may be a new hormone. This method involves acid-alcohol extraction, gel filtration, DEAE-cellulose chromatography, and droplet countercurrent distribution. The peptide contains 36 amino acids, has a molecular weight of 4240 and the isoelectric point if pH 6 to 7. The average amount of avian pancreatic polypeptide extractable from chicken pancreas was 4 mg/100 g of pancreas. The amino acid sequence of the peptide is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2. 相似文献
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Preparative agarose gel electrophoresis for the purification of small organelles and particles 总被引:6,自引:0,他引:6
A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically "downstream," well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellular structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of Mr 142,000, and an ovoid species (70 X 35 nm) whose protein composition is dominated by a polypeptide of Mr 104,000. 相似文献
7.
An immunochemical assay for tubulin subunits is described. The method is applied directly to homogenates of Chlamydomonas reinhardtii solubilized in sodium dodecyl sulfate (Na dodecyl-SO4), and it makes use of a two-dimensional electrophoresis system; the first separation is carried out by Na dodecyl-SO4-polyacrylamide gel electrophoresis and the second by electrophoresis into an agarose gel containing antibodies. Tubulin is precipitated in the form of a "rocket" and the method is made quantitative through the use of cells labeled with [35S]sulfate. The antiserum used in this assay was prepared in rabbits using beta subunit of tubulin purified from Chlamydomonas flagella by two preparative Na dodecyl-SO4-polyacrylamide gel electrophoreses. This antiserum and an antiserum to alpha subunit of tubulin from porcine brain, prepared for comparative study, were extensively characterized. Both antisera show specificity for the polypeptide used as antigen and react with the native dimeric tubulin. The antiserum to beta subunit from Chlamydomonas flagella also forms immunoprecipitates with native brain tubulin and its beta subunit when used at high titer. In contrast, the antiserum to alpha subunit from porcine brain does not cross-react with Chlamydomonas tubulin. The immunochemical assay was applied to Chlamydomonas cells synchronized by a 12-h light/dark cycle. In cells collected during the light period (late G1), after removal of flagella, the content of tubulin is estimated to be 0.3% of total protein. As cells enter the dark period there is a striking increase in tubulin content which reaches a maximum just before cell division. 相似文献
8.
Purification and characterization of 3-dehydroquinate hydrolase and shikmate oxidoreductase. Evidence for a bifunctional enzyme 总被引:6,自引:0,他引:6
L D Polley 《Biochimica et biophysica acta》1978,526(1):259-266
The basis for the physical association of 3-dehydroquinate dehydratase (3-dehydroquinate hydrolyase, EC 4.2.1.10) and shikimate dehydrogenase (shikimate: NADP+ 3-oxidoreductase, EC 1.1.1.25) in higher plants was investigated. The enzymes were extracted from the moss Physcomitrella patens and were purified to homogeneity. Determinations of subunit sizes were made by sodium dodecyl sulfate gel electrophoresis and gel exclusion chromatography in 6 M guanidinium chloride. Results from these studies demonstrate that both enzyme activities are carried out by a single polypeptide. 相似文献
9.
Quantitative determination of overlapped proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis 总被引:1,自引:0,他引:1
A method for resolving an overlapped polypeptide pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis was described. The procedure was essentially a Gaussian fitting using the least squares method, and could resolve more than 20 overlapped components simultaneously. The applicability to overlapped and shouldered patterns was evaluated using practical electrophoretic data with varying amounts of mitochondrial samples. The relative contents of respective polypeptide components gave a good agreement regardless of the loaded amounts. 相似文献
10.
A method is described for extracting proteins and peptides from stained sodecyl sulfate-polyacrylamide gels. Coomassie blue and sodium dodecyl sulfate present in stained gel sections are removed to allow subsequent analysis of the peptides (e.g., amino acid analysis or tryptic digestion and fingerprinting). The method is simple, requires no radioisotopes or special equipment, and can be carried out with a minimum of handling of the sample. The process can be used for samples at the nanomole level with recoveries of 90%. 相似文献
11.
T Oberholzer K H Nierhaus P L Luisi 《Biochemical and biophysical research communications》1999,261(2):238-241
Compartmentalization is one of the key steps in the evolution of cellular structures and, so far, only few attempts have been made to model this kind of "compartmentalized chemistry" using liposomes. The present work shows that even such complex reactions as the ribosomal synthesis of polypeptides can be carried out in liposomes. A method is described for incorporating into 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) liposomes the ribosomal complex together with the other components necessary for protein expression. Synthesis of poly(Phe) in the liposomes is monitored by trichloroacetic acid of the (14)C-labelled products. Control experiments carried out in the absence of one of the ribosomal subunits show by contrast no significant polypeptide expression. This methodology opens up the possibility of using liposomes as minimal cell bioreactors with growing degree of synthetic complexity, which may be relevant for the field of origin of life as well as for biotechnological applications. 相似文献
12.
Purification of a growth factor related to platelet-derived growth factor and a type beta transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors. 总被引:4,自引:0,他引:4
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A J Van den Eijnden-Van Raaij I Koornneef T M Van Oostwaard A Feyen W Kruijer S W De Laat E J Van Zoelen 《The Biochemical journal》1989,257(2):375-382
A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets. 相似文献
13.
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
14.
The polypeptide subunits of cytochrome c oxidase (E.C. 1.9.3.1.) isolated from beef heart which react with 203[Hg]Cl2 have been identified. A rapid and simple method for identifying and numbering the subunits is described which is independent of the type of sodium dodecyl sulphate-polyacrylamide gel system employed to separate them. 相似文献
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In many cases RNA can assume co-existing or meta-stable structures preventing structure determination by chemical mapping. A novel method is described, by which RNA is modified with dimethyl sulphate without shifting the distribution of different structures. The different structures are then separated in native gel electrophoresis, and structure determination by primer extension can be carried out separately for each structure. 相似文献
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A simple method is described for the analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with crossed immunoelectrophoresis. The technique allows the identification of specific proteins and estimation of their molecular weights after the electrophoresis of complex mixtures such as blood plasma. Human ceruloplasmin was estimated to have a molecular weight of 128,000 using this method. Reduction of plasma with 2-mercaptoethanol in the presence of protease inhibitors did not result in dissociation of ceruloplasmin into detectable smaller subunits, suggesting that native ceruloplasmin is composed of a single polypeptide chain. 相似文献
19.
Secretory form of atrial natriuretic polypeptide as cardiac hormone in humans and rats 总被引:4,自引:0,他引:4
K Nakao A Sugawara S Shiono Y Saito N Morii T Yamada H Itoh M Mukoyama H Arai M Sakamoto 《Canadian journal of physiology and pharmacology》1987,65(8):1756-1761
To elucidate the secretory form of atrial natriuretic polypeptide from the atrium, the molecular form of atrial natriuretic polypeptide in the perfusate from the isolated beating rat heart and in plasma taken at the coronary sinus of 10 patients during cardiac catheterization has been investigated using high performance gel permeation chromatography and reverse phase high performance liquid chromatography coupled with radioimmunoassay for atrial natriuretic polypeptide. Atrial natriuretic polypeptide in the perfusate from the rat heart showed a single peak eluting at the position of a low molecular weight form of atrial natriuretic polypeptide, without any detectable amounts of atrial natriuretic polypeptide with high molecular weights. The major component of atrial natriuretic polypeptide in the rat heart perfusate co-migrated with rat alpha-atrial natriuretic polypeptide in reverse phase high performance liquid chromatography. In 9 out of 10 patients atrial natriuretic polypeptide in plasma taken at the coronary sinus revealed a single peak of atrial natriuretic polypeptide emerging at the position of human alpha-atrial natriuretic polypeptide in gel filtration. Only one plasma sample had a small quantity of high molecular weight forms with the predominant low molecular weight form of atrial natriuretic polypeptide. The major component of atrial natriuretic polypeptide in the plasma extract from the coronary sinus was identified with human alpha-atrial natriuretic polypeptide. These results indicate that alpha-ANP, a 28-amino acid polypeptide, is secreted as a cardiac hormone into the coronary blood stream from the atrium. 相似文献
20.
Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis 总被引:7,自引:0,他引:7
A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Sch?gger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected. 相似文献