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Comparison of focusing in buffers and synthetic carrier ampholytes for use in the first dimension of two dimensional polyacrylamide gel electrophoresis
Author(s) -
Burghes Arthur H. M.,
Patel Ketan,
Dunn Michael J.
Publication year - 1985
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150060907
Subject(s) - isoelectric focusing , chemistry , chromatography , polyacrylamide gel electrophoresis , immobilized ph gradient , polyacrylamide , two dimensional gel electrophoresis , resolution (logic) , biochemistry , proteomics , artificial intelligence , polymer chemistry , computer science , gene , enzyme
Buffer focusing using the mixture Polybuffer 47 (Polysciences) has been tested for the separation of proteins under denaturing conditions to assess its potential for use as the first dimension in two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). a relatively simple pattern, with the proteins confined to certain regions of the gel, was observed when [ 35 S]methionine‐labelled fibroblasts were separated both by 2D‐PAGE and by one‐dimensional isoelectric focusing (IEF) using Polybuffer 47. When Polybuffer 47 and synthetic carrier ampholytes were mixed, the latter were also found to concentrate into regions corresponding to those occupied by the proteins. The addition of synthetic carrier ampholytes resulted in separation of proteins within these regions. Diagonal IEF gels of synthetic carrier ampholyte versus buffer focusing showed a “step” pattern, suggesting that buffer focusing resulted in concentration of proteins in particular regions. Diagonal IEF gels using synthetic carrier ampholytes in both dimensions verified that protein‐ampholyte interactions are not a major source of artefacts in such gels. When mixtures of Polybuffer 47 and synthetic carrier ampholytes were used, there was some extension of the pH gradient at both ends. We conclude that Polybuffer 47 alone is not suitable for use in the first dimension of high‐resolution 2D‐PAGE separations.