title: Rho family and Rap GTPase activation assays.
abstract: The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. 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 the upstream Rho or Ras GTPase. Coupling these probes to beads enables extraction of the complex and subsequent quantification of the 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 every small GTPase, analysis of certain members of the Rho and Ras GTPase family is now routinely performed. Here, we describe affinity-based pulldown assays for detection of Rho GTPase (Rac1/2, Cdc42, RhoA/B) and Rap1/2 activity in stimulated neutrophils or macrophages.
authors: Conway Institute, University College Dublin, Dublin, Ireland.
authors: Richard T
authors: RT
authors: Jennings
authors: Ulla G
authors: UG
authors: Knaus
mesh_terms: D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D020668:rap GTP-Binding Proteins; D020741:rho GTP-Binding Proteins