Two‐dimensional analysis of membrane proteins with isoelectric focusing in immobilized pH gradients in the first dimension

Two‐dimensional electrophoresis with isoelectric focusing in immobilized pH gradients in the first dimension was applied to the fractionation of hydrophobic proteins from the plasma membrane of a Streptococcus strain. The detergents incorporated into the first‐dimensional gel slab, especially the zwitterionic ones of the sulfobetaine series, interfere with protein transfer from the first to second dimension by formation of mixed micelles with sodium dodecyl sulfate molecules. In order to reduce their concentration an elution protocol was devised including: (i) fixation for 1 h in 50 % methanol ‐ 12 % acetic acid; (ii) washing with distilled water for 30 min; (iii) equilibration in concentrated Tris buffer; (iv) denaturation in 5 % sodium dodecyl sulfate ‐ 2 % 2‐mercaptoethanol. When counting the number of resolved spots in the protein patterns after two‐dimensional electrophoresis, the relative solubilizing efficiency of different detergents scored as follows: Nonidet P‐40 (NP‐40) > (3‐[3‐cholamidopropyl)dimethylammonio]‐1‐propananesulfonate (CHAPS) > N‐dodecyl‐N, N‐dimethylammonio‐3‐propanesulfonate (sulfobetaine SB 12).