Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients.
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.
Rabilloud
T
T
CEA-Laboratoire de Bioénergétique Cellulaire et Pathologique, UA 2019 DBMS/BECP, Grenoble, France. Thierry@sanrafael.ceng.cea.fr
Adessi
C
C
Giraudel
A
A
Lunardi
J
J
D000818:Animals; D004023:Dictyostelium; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis*; D012995:Solubility; D014404:Tubulin / Q000032:analysis*
D008565:Membrane Proteins; D009687:Nuclear Proteins; D014404:Tubulin