摘要:

AbstractThis study investigates some of the problems encountered in quantitation of proteins in silver stained polyacrylamide gels. Subunit molecular weight, isoelectric point and amino acid content are examined as possible factors for differential staining intensities of proteins. We also describe a procedure for quantitation using atomic absorption spectroscopy, independent of the colors formed upon silver staining. Using this method we show that some proteins such as pepsin bind silver but fail to develop visual color reactions.

ISSN:0173-0835    

DOI:10.1002/elps.1150060802

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

年代:1985

数据来源: WILEY

摘要:

AbstractA negative aurodye for polyacrylamide gels has been developed. After isoelectric focusing in 6 % T polyacrylamide gels, protein fixation and washing, the gel is immersed in 0.01 % HAuCl4containing 0.19 % citric acid and 0.1 % Tween 20, freshly made. The free reactants diffuse within the gel fibers and, upon micelle formation, stain the gel matrix purple, leaving white protein zones in sharp contrast. The sensitivity is of the order of 15–20 ng protein/mm2, matching and even passing that of silver stain (the lowest detection limit of Coomassie Blue being around 300 ng protein/mm2). The mechanism of background stain does not seem to depend on hydrophobic interaction with the gold‐detergent micelle, as polyacrylamide gelled with hydrophobic cross‐linkers has markedly decreased stain‐binding ability. The formation of mixed micelles of gold with non‐ionic, zwitterionic and anionic detergents has bee

ISSN:0173-0835    

DOI:10.1002/elps.1150060803

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

年代:1985

数据来源: WILEY

摘要:

AbstractProtein C (PC), a plasma zymogen of a serine amidase with strong anticoagulant activity, has been purified to homogeneity as shown by sodium dodecyl sulfate electrophoresis. After isoelectric focusing in immobilized pH gradients, PC consists of six isobands, with isoelectric points (pI) between pH 4.8 and 5.1. That all the isobands were PC was confirmed by electroblotting on cellulose nitrate followed by immunoperoxidase staining. Activated PC gives a similar pattern of bands, with pIsca.0.5 units higher. Activated PC was also recognized by the monospecific antibody.

ISSN:0173-0835    

DOI:10.1002/elps.1150060804

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe resolution of continuous flow electrophoresis systems is generally measured by the spread of the sample bands in the direction of the electrophoretic migration. This spreading assumes an electric field that extends uniformly between the two electrodes. This paper evaluates the cross‐section of the sample bands in the plane perpendicular to the flow and shows that the spread in the direction perpendicular to the migration increased significantly with the applied electic field. Concentrated samples of monodisperse latex particles and vinyltoluene T‐butylstyrene particles in sample buffers of different electrical conductivities were used to map the shape of the sample bands relative to the zero electric field case. As the electric field was applied, the sample band spread from an initial diameter of only one‐third the chamber thickness until it approached the chamber walls where electroosmosis significantly reduced the resolution of separation. It can be shown, however, that it is possible to minimize these distortions by careful sample preparation and experiment d

ISSN:0173-0835    

DOI:10.1002/elps.1150060805

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

年代:1985

数据来源: WILEY

摘要:

AbstractFor proper molecular weight determination in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the protein should bind the same amount of SDS. High ionic strength had previously been shown to reduce the amount of SDS bound to proteins, suggesting that high salt concentrations in the samples may interfere with molecular weight determination. We examined the effects of NaCl from 0 to 1 M in the samples on molecular weight determination of proteins in SDS‐PAGE. In our conditions of electrophoresis, the retardation coefficients (KR), free mobilities (Yo) andRfvalues for proteins (Mr20 000–66 000) are not affected by NaCl in the samples up to 0.8 M. Therefore molecular weight of these proteins can be determined by both the Ferguson plot andRfvs.logMrmethods in the presence of NaCl in the samples up to 0.8 M and compared with standard protein samples in the absence of NaCl. TheRf,KRandYovalues for phosphorylase (Mr97 400) are affected by NaCl from 0.4 M or higher in the samples. Preliminary results show that increasing sodium phosphate, pH 7.0 from 0.01 M to 0.1 M in the sample affect molecular weight determinations. The effects of phosphate are greater in the Ferguson plot than in theRfvs.logMrplot procedure. In the latter method, the effects are dependent on the acrylamide gel concentrations and on the size of the proteins. We conclude that the molecular weight of proteins (Mr20 000 to 97 000) can be determined in the presence of high salt concentration in the sample if the unknown and standard protein samples contain the same amount of salt and buff

ISSN:0173-0835    

DOI:10.1002/elps.1150060806

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

年代:1985

数据来源: WILEY

摘要:

AbstractA new technique, multiple crossed immunoelectrophoresis on agarose‐coated GelBond film, is described. A sheet of GelBond with agarose areas containing a single or different antisera can be used for the simultaneous analysis of eight samples. Antigens were separated in the first dimension (i) in an inert medium according to net charge (cellulose acetate membrane elecrophoresis), (ii) in a sieving medium according to charge and molecular size (ultrathin‐layer polyacrylamide gradient gel electrophoresis), or (iii) in a pH gradient according to differences in isoelectric points (ultrathinlayer isoelectric focusing). By using these methods in the first dimension, resolution is improved over that in conventional agarose electrophoresis. The first dimensional strips are easily transferred to the second dimensional antibody‐containing agarose. Use of the GelBond film allows easy handling and convenient storage. Multiple crossed immunoelectrophoresis is a versatile method for rapid analysis of antigenic variab

ISSN:0173-0835    

DOI:10.1002/elps.1150060807

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

年代:1985

数据来源: WILEY

摘要:

AbstractFour two‐dimensional electrophoresis methods, each incorporating different protein denaturing/dissociating conditions, have been used to detect changes in rat serum proteins following exposure to dimethylformamide. The methods were evaluated on the basis of (i) their ability to detect changes, (ii) the resolution achieved and (iii) the response of resolution to the use of narrow Ampholine pH ranges in the first dimensional isoelectric focusing gels. All methods reveal minor but reproducible protein changes in response to dimethylformamide. However, high resolution two‐dimensional electrophoresis of the completely dissociated polypeptides reveals most changes and the resolution of this method is most improved by the use of Ampholines of narrow pH range. In general, the number of serum components detected and the resolution achieved is related to the severity of the protein denaturing/dissociating conditions employed. The significance of this observation in relation to others factors likely to influence the choice of two‐dimensional method is disc

ISSN:0173-0835    

DOI:10.1002/elps.1150060808

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

年代:1985

数据来源: WILEY

摘要:

AbstractSerum specimens from local Whites, Blacks and Amerindians were phenotyped for the B subunit of Factor XIII. Isoelectric focusing of desialylated samples on agarose gels at a pH 4–7 gradient followed by an immunoblotting technique was used for band identification. Gene frequencies for the three common alleles in Whites, F XIII*1, F XIIIB*2 and F XIIIB*3 were 0.776, 0.088 and 0.136, respectively, and were consistent with reports on this genetic marker system in European Whites. The Blacks and Amerindians were clearly differentiated from the Whites with F XIIIB*1,2 and 3 frequencies of 0.286, 0.635 and 0.079, respectively, for Blacks and 0.500, 0.034 and 0.466 for Amerindians. Based on the available population data on Factor XIIIB, this genetic system should prove to be a valuable marker for anthropological genetics and parentage testin

ISSN:0173-0835    

DOI:10.1002/elps.1150060809

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

年代:1985

数据来源: WILEY

摘要:

AbstractTwo narrow immobilized pH gradients (pH 4.35–4.85 and pH 4.40–4.80) were formulated by linear interpolation from standard 1 pH unit ranges. Both gradients proved excellent for alpha‐1‐antitrypsin phenotype separation. Gel casting was simplified and the properties of the gels during the run improved by replacing glycerol with sucrose in the polymerization so

ISSN:0173-0835    

DOI:10.1002/elps.1150060810

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe staining of proteins in polyacrylamide gels using Gelcode® silver staining can be enhanced approximately tenfold by fixing with glutaraldehyde. The glutaraldehyde results in the formation of a protein‐aldehyde complex. This complex retards the diffusion of low molecular weight proteins from the gel and increases staining sensitivity by increasing the amount of metallic silver deposited within the protein‐aldehyde com

ISSN:0173-0835    

DOI:10.1002/elps.1150060811

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

年代:1985

数据来源: WILEY