Nonradioactive determination of Ras-GTP levels using activated ras interaction assay

Publisher Summary This chapter describes the nonradioactive determination of Ras- guanosine-5'-triphosphate (GTP) levels using activated ras interaction assay. In their activated, GTP-bound forms the Ras proteins interact with a variety of intracellular effector proteins to initiate signaling pathways that contribute to cell proliferation, differentiation, or death, depending on cellular context. The level of Ras activation (the fraction of total Ras molecules in the GTP-bound form) and the duration of Ras activation (the length of time a given fraction is in the GTP-bound form) are controlled by the opposing actions of exchange factors that stimulate guanosine-diphosphate (GDP) for GTP exchange and GTPase-activating proteins (GAPs), which stimulate the intrinsic GTPase activity of Ras. The first generation of Ras—GTP assays directly measured the ratio of GTP to GDP bound to Ras extracted from cells. If trying to detect a low level of Ras activation it may be necessary to optimize assay conditions using normally activated—that is, nonmutated, Ras.