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.