Mapping small GTP‐binding proteins on high‐resolution two‐dimensional gels by a combination of GTP binding and labeling with in situ periodateoxidized 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.