Membrane protein analysis by isoelectric focusing in immobilized pH gradients

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Abstract
Non‐ionic detergents (NP‐40, Triton X‐100) have poor solubilizing properties on membrane proteins, as demonstrated by separating NP‐40‐extracted proteins by isoelectric focusing also in the absence of carrier ampholytes (Ampholine), i. e. in immobilized pH gradients (IPG). Addition of 4% Ampholine to the sample and to the IPG gels results in an increase in membrane solubility, from 40% in NP‐40 alone to 60% in NP‐40 with 4% Ampholine, when both extractions were in the absence of 8 M urea. If the latter solvent is used, these values are increased to 85% for the former and to 100% for the latter extraction conditions. In presence of carrier ampholytes sharp bands are obtained on isoelectric focusing. It is hypothesized that neutral detergents in presence of Ampholine form mixed micelles, mimicking the properties of natural zwitterionic surfactants.