The GTPase-Glo Assay assesses the activities of GTPases, GAPs and GEFs, which are components of the GTPase cycle, by detecting the amount of GTP remaining after GTP hydrolysis in a GTPase reaction. The remaining GTP is converted to ATP using the GTPase-Glo Reagent, and the ATP is then detected using a proprietary thermostable luciferase (Ultra-Glo Recombinant Luciferase) and luciferin substrate to produce bioluminescence. The kit contains optimized reaction buffers, GTPase/GAP Buffer and GEF Buffer, for performing GTPase and GAP reactions and GEF reactions, respectively. These two buffers primarily differ in their Mg2+ content, which is critical for the nucleotide loading and unloading of the GTPase, thereby affecting the GTPase cycle. With the GTPase-Glo Assay, you can measure intrinsic GTPase activity, GAP-stimulated GTPase activity, GAP activity and GEF activity. GTPase, GAP and GEF activity is inversely correlated to the amount of light produced. A highly active GTPase hydrolyzes more GTP, reducing the amount of ATP produced from GTP and reducing light output. A less active GTPase hydrolyzes less GTP, leaving a larger amount of GTP to be converted to ATP and producing more light.
