Methods in Molecular Biology book series (MIMB, volume 1124)AbstractThe 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.KeywordsRho GTPases Rac Cdc42 Rap Affinity-based GTP activity probe PBD assay RBD assay Pulldown assay This is a preview of subscription content, log in to check access.Springer Nature is developing a new tool to find and evaluate Protocols.Learn moreNotesAcknowledgmentsThis work was supported by grants from Science Foundation Ireland and the Health Research Board.References1.Dinauer MC (2003) Regulation of neutrophil function by Rac GTPases. Curr Opin Hematol 10:8–15PubMedCrossRefGoogle Scholar2.Bokoch GM (2005) Regulation of innate immunity by Rho GTPases. 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Trends Cell Biol 21:615–623PubMedCrossRefGoogle ScholarCopyright information© Springer Science+Business Media, LLC 2014Authors and AffiliationsRichard T. Jennings1Ulla G. Knaus11.Conway InstituteUniversity College DublinDublinIrelandAbout this protocol Cite this protocol as: Jennings R.T., Knaus U.G. (2014) Rho Family and Rap GTPase Activation Assays. In: Quinn M., DeLeo F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology (Methods and Protocols), vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_6 First Online 17 January 2014 DOI https://doi.org/10.1007/978-1-62703-845-4_6 Publisher Name Humana Press, Totowa, NJ Print ISBN 978-1-62703-844-7 Online ISBN 978-1-62703-845-4 eBook Packages Springer Protocols