Premium
Very alkaline immobilized pH gradients for two‐dimensional electrophoresis of ribosomal and nuclear proteins
Author(s) -
Görg Angelika,
Obermaier Christian,
Boguth Günther,
Csordas Adam,
Diaz JeanJacques,
Madjar JeanJacques
Publication year - 1997
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.1150180306
Subject(s) - immobilized ph gradient , electrophoresis , isoelectric focusing , chromatography , chemistry , ribosomal protein , capillary electrophoresis , polyacrylamide gel electrophoresis , isoelectric point , hela , gel electrophoresis , biochemistry , ribosome , cell , enzyme , rna , gene
Basic proteins normally lost by the cathodic drift of carrier ampholyte focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two‐dimensional (2‐D) electrophoresis under equilibrium conditions using immobilized pH gradients (IPGs) 4–10 and 6–10 using a previously published protocol (Görg et al., Electrophoresis 1988, 9, 531–546). In the present study we have extended the pH gradient to pH 12 with IPGs 8–12, 9–12 and 10–12 for the analysis of very basic proteins. Different optimization steps with respect to pH engineering, gel composition and running conditions, such as substitution of acrylamide by dimethylacrylamide and addition of isopropanol with and without methylcellulose to the IPG rehydration solution (in order to suppress the reverse electroosmotic flow) were necessary to obtain highly reproducible 2‐D patterns of ribosomal proteins from HeLa cells and mouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under steady‐state conditions. Due to the selectivity of isoelectric focusing in IPG 9–12, where the more acidic proteins abandon the gel, the tedious procedure of nuclei preparation prior to histone extraction can be omitted.