Rac and Rap GTPase activation assays.

Abstract

The detection of Ras superfamily GTPase activity in neutrophils is important when studying signaling events elicited by various ligands and cellular processes. Substantial progress in monitoring GTPase activation has been made in recent years by the development of high-affinity probes for small GTPases. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione-S-transferase (GST). Such domains bind preferentially to the GTP-bound form of specific Rho or Ras GTPases. Coupling these probes to beads enables extraction of the complex and subsequent quantification of active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for many small GTPases, analysis of certain members of the Rho and Ras GTPase family are now routinely performed. Here, we describe affinity-based pull-down assays for detection of Rac/Cdc42 and Rap activity in stimulated neutrophils.