A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.