Mapping small GTP-binding proteins on high-resolution two-dimensional gels by a combination of GTP binding and labeling with in situ periodate-oxidized GTP.
We compared two approaches to identify and map small GTP-binding proteins in combination with high-resolution two-dimensional (2-D) gel electrophoresis. The first approach involved direct GTP ligand binding after a renaturing transfer onto nitrocellulose. In the second, affinity labeling with in situ periodate-oxidized GTP was used in permeabilized cells (Peter, M. E., She, J., Huber, L. A. and Terhorst, C. Anal. Biochem. 1993, 210, 77-85). Analysis by 2-D gel electrophoresis revealed a number of distinct intracellular small GTP-binding proteins in Madine-darby canine kidney strain II cells (MDCKII). Using specific antibodies the electrophoretic coordinates of rab4, rap1a/b, and rap2 were identified for native as well as for crosslinked GTPases. These methods allow the identification of small GTP-binding proteins in total cell lysates and purified subcellular fractions, providing excellent markers throughout the course of differentiation and development.
Cell Biology Program, European Molecular Biology Laboratory, Heidelberg.
L A
LA
Huber
M E
ME
Peter
D000818:Animals; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D020558:GTP Phosphohydrolases / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D010504:Periodic Acid / Q000737:chemistry*
D010504:Periodic Acid; D006160:Guanosine Triphosphate; C009288:metaperiodate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins