VERY ALKALINE IMMOBILIZED PH GRADIENTS FOR 2-DIMENSIONAL ELECTROPHORESIS OF RIBOSOMAL AND NUCLEAR PROTEINS

Basic proteins normally lost by the cathodic drift of carrier ampholyt e focusing, or separated by NEPHGE with limited reproducibility, could be well separated by two-dimensional (2-D) electrophoresis under equi librium conditions using immobilized pH gradients (IPGs) 4-10 and 6-10 using a previously published protocol (Gorg et al., Electrophoresis 1 988, 9, 531-546). In the present study we have extended the pH gradien t to pH 12 and IPGs 8-12, 9-12 and 10-12 for the analysis of very basi c proteins. Different optimization steps with respect to pH engineerin g, gel composition and running conditions, such as substitution of acr ylamide by dimethylacrylamide and addition of iso-propanol with and wi thout methylcellulose to the IPG redydration solution (in order to sup press the reverse electroosmotic flow) were necessary to obtain highly reproducible 2-D patterns of ribosomal proteins from HeLa cells and m ouse liver. Histones from chicken erythrocyte nuclei as well as total cell extracts of erythrocytes were also successfully separated under s teady-state conditions. Due to the selectivity of isoelectric focusing in IPG 9-12, where the more acidic proteins abandon the gel, the tedi ous procedure of nuclei preparation prior to histone extraction can be omitted.