Solubilization of plasma membranes in anionic, non-ionic and zwitterionic surfactants for iso-dalt analysis: a...

A critical study has been made of the solubilizing properties of anionic, nonionic and zwitterionic surfactants to be used in the first dimension of two-dimensional isoelectric focusing-sodium dodecyl sulphate (IF-SDS) maps. Excess of SDS is a powerful solubilizing agent, but prevents proteins from entering the IF gel. Nonidet P-40 (NP-40)-urea mixtures are mediocre solubilizing agents, but are compatible with the IF dimension. Zwitterionic detergents (zwittergents) appear to exhibit a well balanced solubilizing power and are able to generate sharp two-dimensional maps, with round spots and minimal vertical and horizontal streaking. SB3-12 and SB3-14 appear to have the best solubilizing properties; shorter surfactants (SB3-8) exhibit a poor solubilization efficiency, while longer detergents (SB3-16) bind too strongly to hydrophobic regions in proteins. The random performance of non-ionic detergents has been attributed to their ability to form mixed micelles with the carrier ampholytes used in the IF step: depending on the relative ratio of NP-40 to Ampholines, different types of micelles would be formed, which, when reaching appropriate stoichiometries and charge densities, would mimic the behaviour of natural zwittergents. Acceptable two-dimensional maps can be obtained when the sample is lysed in limiting amounts of SDS (SDS:protein = 1:3), suggesting that excess of free SDS in solution is deleterious to the IF process.