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Factors affecting the range of pH gradients in the isoelectric focusing dimennsion of two‐dimensional gel electrophoresis: The effects of reservoir electrolytes and loading procedures
Author(s) -
Chambers John A. A.,
Innocenti Francesco Degli,
Hinkelammert Katharina,
Russo Vincenzo E. A.
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.1150060707
Subject(s) - isoelectric focusing , electrolyte , chemistry , chromatography , polymerization , electrophoresis , immobilized ph gradient , isoelectric point , gel electrophoresis , analytical chemistry (journal) , two dimensional gel electrophoresis , polymer , organic chemistry , electrode , biochemistry , enzyme , proteomics , gene
We have examined methods for broadening and stabilising pH gradients used in the first dimension of two‐dimensional gel electrophoresis. The replacement of typical strong electrolytes with weak electrolytes as reservoir anolyte and catholyte allows the generation of broad (3.5 pH unit) gradients that are stable for at least 28 000 volt × hours (Vh). Protein patterns form within 10 000 Vh are stable throughout the subsequent focusing. Large quantities of protein, up to 6 mg, can beloaded onto such gels by mixing samples into the gel prior to polymerisation. We found no evidence for extensive modification of proteins because of exposure to the polymerisation reaction. The pH gradient formed in strong electrolytes in the typical O'Farrell gel system ( J. Biol. Chem. 1975, 250 , 4007–4021) could also be broadened if the samples were again mixed into the gel before polymerisation. This last effect appears to be due to an effect of the sample buffer used upon end loading of samples.