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A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale
Author(s) -
Bjellqvist Bengt,
Pasquali Christian,
Ravier Florence,
Sanchez JeanCharles,
Hochstrasser Denis
Publication year - 1993
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.11501401209
Subject(s) - immobilized ph gradient , reproducibility , chemistry , chromatography , urea , electrophoresis , range (aeronautics) , resolution (logic) , concentration gradient , nonlinear system , analytical chemistry (journal) , amino acid , materials science , computer science , enzyme , biochemistry , isoelectric focusing , artificial intelligence , composite material , physics , quantum mechanics
A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two‐dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on p K values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10–25°C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.