ISSN:0173-0835    

DOI:10.1002/elps.1150030102

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

年代:1982

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150030103

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe use of silver for detection of protein in polyacrylamide gels is becoming widespread. An effort has been made to develop a silver stain which minimizes the time to perform the stain, the amount of silver used, and the complexity of the procedures, and which maximizes the sensitivity. This stain is almost 200‐fold more sensitive than the Coomassie Brilliant Blue R‐250 stain. This silver stain procedure has been shown, with eight purified proteins, to generally be linear over a 40‐fold range in concentration, between 0.005 ng/mm2to 2.0 ng/mm2. For greater sensitivity, a recycling procedure has been developed. This procedure is capable of amplifying trace proteins which could not be visualized with previous silver stain techniques. Analysis of the kinetics of image development revealed that the silver ion was the limiting factor in image formation. By recycling the gel through silver nitrate solutions, silver ions are replenished in the gel, permitting amplification of further details when the gel is treated with a developing solution. A number of problems inherent in silver stain detection of proteins in polyacrylamide gels are discussed with suggested rem

ISSN:0173-0835    

DOI:10.1002/elps.1150030104

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

年代:1982

数据来源: WILEY

摘要:

AbstractA simplified, inexpensive silver stain for proteins in polyacrylamide gels also stains DNA with a sensitivity of 1 ng (<0.03 ng/mm2). Sensitivity of detection is superior to ethidium bromide and, for 11 double‐stranded fragments of ϕX174 DNA, staining is linear with respect to concentration. Surprisingly, the slope of staining is similar for pieces of DNA larger than 300 base pairs, but is greater for fragments of smaller size. Staining of DNA with silver is a practical alternative to visualization with ethidium bromide and may serve as a tool for elucidating the basis of silver staini

ISSN:0173-0835    

DOI:10.1002/elps.1150030105

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

年代:1982

数据来源: WILEY

摘要:

AbstractA method for protein staining after isoelectric focusing (IEF) in polyacrylamide gel is described in which the total time for fixation, staining and complete gel background destaining is reduced to 3–5 min. This is achieved by (i) staining dry gels on silanized supports instead of hydrated gels, (ii) reducing gel thickness to 50 μm, (iii) using Servalyt carrier ampholytes with good destaining properties, and (iv) using dyes with low affinity for carrier ampholytes, namely Serva Violet 17 or 49, facilitating destaining of gel background. The sensitivity of staining is 100–200 ng protein/ mm2over a 10 cm separation distance and 20–30 ng protein/ mm2over 3 cm gels (miniature focusing). Addition of 5–20 % methyl acetate to the staining or destaining solution reduces the sensitivity of staining significantly. Most effective fixation of proteins in 50 μm gels is achieved with 20% trichloroacetic acid. In solutions of alcohol and acetic acid, appreciable amounts of proteins are leached after incubation for 5–20 min. Paraformaldehyde, malondialdehyde or glutaraldehyde (5–10 %) exhibit no fixative effect on proteins in 50 μm gels when used directly or after precipitation of the proteins with trichloroacetic acid. The selective leaching of proteins under a variety of experimental conditions shows that adequate fixation is particularly important in work with basic, low molecular weight and/or hydrop

ISSN:0173-0835    

DOI:10.1002/elps.1150030106

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

年代:1982

数据来源: WILEY

摘要:

AbstractUntreated and processed gel plates of polyacrylamide (PAA) gradient gels were cut into strips perpendicularly to their length, and the wet and dry matter of the sections was determined. In untreated gels the apparent dry matter, as well as the relative dry matter, are a linear function of the gel length. In processed gels, however, only the apparent gel concentration increases linearly with the gel length, whereas the relative dry matter increases linearly with the square root of the gel length. The %T (content in polyacrylamide) was calculated from the apparent dry matter. The gel gradients used were found to be linear with respect to %T. Six different calibration proteins were run and their time‐dependent migration distances determined. For each protein a linear relationship exists between the square root of its migration distance (√D) and the logarithm of the electrophoretical duration (log t). In this relation √D can be substituted by the square root of the PAA concentration\documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document}or by the relative dry matter (TM(%)). The resulting straight lines seem to intersect near the zero time of electrophoresis and the starting concentration of the gradient. At any time during electrophoresis (t» O), the square root of the migration distance (√D) of the calibration proteins and the logs of their Stokes' radii (log Rs), or the logs of their molecular weights (log MW), are correlated linearly. In this function, √D may be replaced by either\documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document}or TM (%). It is shown that this correlation is not subject to time, although the constants are changed during the process of electrophoresis. The slopes of the lines and the duration of the electrophoresis (at a constant voltage) are related by a hyperbolic function. Regression lines obtained for different times during electrophoresis intersect in the range of the starting gel concentration. A more general equation is formulated which states ‐ within certain experimental limitations — the size of proteins, their migration distances, and the time of e

ISSN:0173-0835    

DOI:10.1002/elps.1150030107

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

年代:1982

数据来源: WILEY

摘要:

AbstractUnder certain conditions in polyacrylamide gradient gel electrophoresis (PAGGE), a linear correlation between the logarithm of the size of calibration proteins (log MW or log Rs) and the square root of their migration distance (√D) can be observed; slope and intercept of the calibration curve depend on the duration of electrophoresis; linearity, however, is maintained over a wide range (4‐60 h, 200 V) (Rothe and Purkhanbaba, Electrophoresis 1982, 3, 33–42.) Using this method the reaction of plant isozyme systems penetrating a linear polyacrylamide (PAA) gradient gel was investigated: lactate dehydrogenase (LDH) from potato tubers behaves similarly to animal calibration proteins. The enzyme exhibits 3 subbands with molecular weights of 144 000, 150 000 and 156 000 (± 1.7 %), respectively. The enzyme system “shikimate oxidoreductase” (SORase) behaves significantly different: during PAGGE it splits into 2 major bands (∼60 000 and ∼110 000, respectively) consisting of several subbands. The results of our experiments show that SORase is present in vivo as a complex system. We believe that this complex is partly indentical with the so‐called pre‐

ISSN:0173-0835    

DOI:10.1002/elps.1150030108

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

年代:1982

数据来源: WILEY

摘要:

AbstractA device commonly used for cell electrophoresis is the cytopherometer. Its application in an immunological tumor test, however, pointed out many shortcomings and instabilities. In order to use cytopherometers in cellular immunology, a procedure was worked out to stabilize this instrument. By enlarging the electrode chambers, using a simple and efficient buffer in all compartments, coating the electrophoresis chamber, and by following a set of electrophoretic parameters (current, temperature,etc.), an intraassay variance of 1.8 % and an interassay variance of approximately 1 % were reached.

ISSN:0173-0835    

DOI:10.1002/elps.1150030109

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

年代:1982

数据来源: WILEY

摘要:

AbstractPlasma from normal male and female rats was analyzed, after removal of albumin by affinity chromatography, by two‐dimensional electrophoresis. The results demonstrated gender differences in two plasma proteins with apparent molecular weights of 70 000 and 84 000 and isoelectric points ranging from 6.2 to 6.4, and 5.1 to 5.5, respectively. Both proteins were found to be more concentrated in the plasma minus albumin (P‐alb) fraction of females than males in both Wistar and Sprague‐Dawley rats. The 70 000 protein is serum albumin which was more completely removed from male plasma than female plasma. A possible mechanism responsible for this difference is discussed. The results support the existence of “maleness” and “femaleness” in blood protein pat

ISSN:0173-0835    

DOI:10.1002/elps.1150030110

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

年代:1982

数据来源: WILEY

摘要:

AbstractAn improved isoelectric focusing (IEF) technique has been developed and used to detect the erythrocyte galactose‐1‐phosphate uridyl transferase isoenzymes. The normal enzyme was resolved by IEF in four bands with isoelectric points (pI) between 5.80 and 6.08. Numerous samples, classified according to electrophoresis as Duarte or Los Angeles homozygotes, showed the same IEF pattern composed of four bands with pI's between 5.60 and 5.90. The Duarte‐normal and Los Angelesnormal heterozygotes gave an IEF pattern of 4‐5 bands (pI 5.60‐6.08), with relative intensities that varied according to their

ISSN:0173-0835    

DOI:10.1002/elps.1150030111

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

年代:1982

数据来源: WILEY