ISSN:0173-0835    

DOI:10.1002/elps.1150070102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractAn improved procedure is described for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis (CIE). The method involves detection of immunoprecipitates by autoradiography of CIE gels dried onto filter paper. This modification allows selected segments of immunoprecipitate arcs to be excised with a high degree of precision. Radiolabeled antigens are extracted from excised precipitates by incubation at 60 °C in Laemmli sample buffer, and polypeptides are visualized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis performed in conjunction with autoradiography or fluorography. Protein antigens of the bacterial outer membrane are shown to retain (in part) their properties of heat‐modifiability following drying and extraction, thus facilitating their identification. The procedure is applied to the analysis of fourteen membrane‐associated antigens ofEscherichia coliand results in the identification of theompF/Cand theompAgene products, and the resolution of two novel heterooligomeric outer membrane protein antigens. The polypeptide composition of four previously uncharacterized inner membrane antigens is also established. In addition, six antigens which had been characterized by other unrelated methods as common protein antigen, β‐galactosidase, adenosine‐5′‐triphosphatase, dihydrolipoyl dehydrogenase, D‐lactate dehydrogenase and thelppgene product (the Braun lipoprotein) are shown to possess polypeptide profiles which confirm the initial identification. Evidence is also presented to support the thesis that the bound form of thelppgene product can associate with other proteins of t

ISSN:0173-0835    

DOI:10.1002/elps.1150070103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractA new approach to isoelectric focusing in polyacrylamide gels is described, based on the use of rehydratable gels which in dry form could be stored for extended periods and which prior to use were rehydrated with solutions of any composition. Ultrathin 60–240 μm polyacrylamide gels, with different composition (5 % T, 3 % C; 3 % T, 4 % C; 3 % T, 20 % C), were polymerized under well‐standardized conditions and, after polymerization, washed exhaustively with distilled water to remove any unreacted monomers, catalysts or soluble polymers. The washed gels were impregnated with suitable additives, before drying, to preserve gel functionality on storage. Polyol compounds, such as glycerol, sorbitol and dextran, as well as synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, were the most efficient additives when incorporated into the gel in a concentration of 1–10 %, either as single substances or in different combinations. Prior to isoelectric focusing the dry gels were rehydrated to the original gel volume with a solution of carrier ampholytes, in some experiments with added separators or urea. Kinetic studies have shown rehydration, depending on gel thickness, to be complete within a few minutes. Isoelectric focusing in rehydratable gels was consistently more reproducible than in wet gels by such criteria as regularity of patterns and coalescence of marker proteins, including ferritin, applied at different positions. Rehydratable gels tolerated higher field strengths at the final stage of isoelectric focusing, with typical values of 500–900 V/cm and thus, at a given volt X hour product, equilibrium focusing could be attained in a shorter time, with improved resolution and sharper zones. Ultrathin‐layer isoelectric focusing in rehydratable gels proved insensitive to high salt concentrations (up to 0.5M) of the samples. Rehydratable gels represent a new generation of gels, excelling over the traditional wet gels by better standardized properties, convenient handling and unsurpassed f

ISSN:0173-0835    

DOI:10.1002/elps.1150070104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe method presented in this article was developed specifically to be used for the electrophoretic separation of biological cells. This method uses low cost materials and ordinary clinical laboratory electrophoresis equipment. Results are reasonably fast; time of migration was 20 min using a constant current setting of 6 mA for a single 3 × 4 inch gel tray. The use of SeaPlaque agarose sol at 40 °C allows the cells to migrate freely, while the solidification of the agarose at the end of the migration period fixes the cells in place on the plate. Samples can be taken from any position on the plate while the sol is still liquid. With this method, differences in rate of migration of erythrocytes from selected species of mammals were demonstrated. The method was developed using erythorcytes for samples and without staining of gels or prestaining of samples. Sample sizes of 2000 to 4000 cells gave easily visible bands. Band of cells from some species migrated as much as 40 mm. Separations were done in polystryrene gel tryays. This technique will be tested in the near future for the separation of the various types of blood cells and for separating other cell mixtures,e. g., bacteri

ISSN:0173-0835    

DOI:10.1002/elps.1150070105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractLiver proteins of male C57BL/6T mice treated with 0, 50, or 250 mg/kg Aroclor 1254 were analyzed by high‐resolution two‐dimensional (2‐D) electrophoresis. The resulting patterns were processed using a computerized image analysis system and quantitative data selected for a total of 150 protein spots. On the basis of an analysis of liver proteins form five animals in each treatment group, we found 31 proteins that showed quantitative differences attributable to treatment with chlorinated hydrocarbons at a high level of statistical significance. One of the altered proteins appears to be Mitcon:2, a heat‐shock sensitive mitochondrial matrix polypeptide; another appears likely to be microsomal cytochromeb5. The results indicate that quantitative 2‐D protein mapping may reveal much more detail regarding thein vivoeffects of toxic xenobiotics than has previously been available, and thus allow a more informative approach to the testing of toxic

ISSN:0173-0835    

DOI:10.1002/elps.1150070106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThis report describes a method ofin vitrolabelling of proteins designed for subsequent analysis of the sample by two‐dimensional electrophoresis. As much as 106cpm can be incorporated in 5 μg of proteins. As the reagent reacts with the amino groups, virtually all the proteins are labelled. Thus, this method is far more sensitive than silver staining and may prove useful when extremely low amounts of sample are to be analys

ISSN:0173-0835    

DOI:10.1002/elps.1150070107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe level of genetic variation revealed by two‐dimensional electrophoresis of proteins from seedlings of two wheat lines strongly depends on the technical procedures. Improvements in extraction and electrophoresis procedures relative to earlier experiments on the same material led to a significant increase in the genetic variation revealed: 15.2 % instead of 6.7 % of the spots were genetically variable. The improved procedure is based on (i) precipipation of proteins from wheat seedlings with trichloroacetic acid and acetone, (ii) solubilization of the proteins with a solution containing urea, potassium carbonate and sodium dodecyl sulfate, (iii) isoelectric focusing in an optimized pH gradient, obtained with a mixture of carrier ampholytes (Pharmalyte and Servalyt), and (iv) running elecrophoresis in the second dimension on gels with increased surfac

ISSN:0173-0835    

DOI:10.1002/elps.1150070108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe interpretation of nucleic acid sequences on polyacrylamide gels is often made difficult due to “smiles”. We have devised a simple modification for commonly used electrophoresis systems to eliminate this prob

ISSN:0173-0835    

DOI:10.1002/elps.1150070109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractLateral streaking or bending in vertical one‐dimensional slab gel electrophoresis can be completely avoided if a comb‐gel made up in electrophoresis buffer is placed on top of the stacking gel or sample

ISSN:0173-0835    

DOI:10.1002/elps.1150070110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150070111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY