Abstract B44: GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities

Abstract

Abstract It is well known that GTPases of the Ras superfamily play major roles in cell functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. We have developed a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GAP and GEF. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP. By manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The GTPase/GAP/GEF-Glo™ assay system enables monitoring of GTPase-, GAP-stimulated GTPase-, GAP- and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening. Citation Format: Subhanjan Mondal, Said A. Goueli. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B44. doi: 10.1158/1557-3125.RASONC14-B44