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Publisher Summary The major GTPase-activating protein (GAP) for Ras, p120 Ras–GAP, was the first protein to be identified that could modulate the nucleotide state of p21 Ras. The use of an in situ detection method has proved to have a number of advantages over more conventional GAP assays, including (1) many extracts (8 per minigel) can be analyzed and compared simultaneously, (2) sample handling time is minimal (with less chance of protein degradation) because tissues extracts are immediately denatured and analyzed, (3) on the basis of their sizes, all major GAPs can be analyzed without the need for specific reagents, (4) potential GAP-inhibitor proteins present in the extracts are dissociated during sodium dodecyl sulfate–polyacrylamide electrophoresis, and (5) filters can be sequentially reprobed with different p21 substrates, thus giving information about the p21 specificity of each GAP band. The chapter describes the labeling of recombinant p21 proteins.

Publisher Summary This chapter describes a method to detect a new family of Rho p21 interacting proteins. It also describes the use of the assay for expression screening and in the purification of an abundant Cdc42 and Rac1 target, which is the brain serine/threonine kinase p65-PAK. The GTPase-activating protein (GAP) overlay technique not only detects tissue levels of GAPs after sodium dodecyl sulfate (SDS) electrophoresis but also detects GTPase-inhibitory proteins. For these proteins to exert such an effect on the intrinsic GTPase activity of p21, they must remain bound (that is, they cannot act in a catalytic manner as do GAPs). After autoradiography of the nitrocellulose overlay filter, which holds the image of the nucleotide state of the test p21 within the original filter, some of these proteins can be observed as signals darker than the background. The process of detection of SDS–polyacrylamide-fractionated GTP-p21-binding proteins is described in the chapter.