Improvement of the solubilization of proteins in two‐dimensional electrophoresis with immobilized pH gradients

TLDR

High‑resolution two‑dimensional gel electrophoresis separates membrane and nuclear proteins of poor solubility, but isoelectric focusing with immobilized pH gradients causes severe quantitative losses despite high resolution. The study aims to improve protein solubility in 2‑D electrophoresis by employing denaturing solutions with detergents and chaotropes. The authors used denaturing solutions comprising various detergents and chaotropes to enhance solubility. The optimal mixture of urea, thiourea, and nonionic/zwitterionic detergents improved solubilization of microsomal, nuclear, and aggregation‑prone tubulin proteins.

Abstract

Abstract Membrane and nuclear proteins of poor solubility have been separated by high resolution two‐dimensional (2‐D) gel electrophoresis. Isoelectric focusing with immobilized pH gradients leads to severe quantitative losses of proteins in the resulting 2‐D map, although the resolution is usually high. Protein solubility could be improved by using denaturing solutions containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and detergents (both nonionic and zwitterionic). The usefulness of thiourea‐containing denaturing mixtures is shown for microsomal and nuclear proteins as well as for tubulin, a protein highly prone to aggregation.