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Bioluminescent Assay for GTPases Allows Measurement of GTPase, GAP and GEF Activities

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins results in in serious pathological conditions. Targeting GTPases and their regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay (GTPase-Glo) system to analyze these proteins in a simple, convenient “add-mix-read” format.

The assay consists of optimized reaction buffers that allow continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and bioluminescent detection of the ATP. We show that the assay is sensitive and robust when analyzing for analyzing intrinsic GTPase- activity, GAP-stimulated GTPase- activity, GAP- activity and GEF- activity. The assay has minimal false hits when tested for compound interference using the LOPAC library (library of pharmacologically active compounds) indicative of the robustness in identifying small molecule modulators of GTPase using high throughput screening