Kras (G12V) Nucleotide Exchange Assay Kit (384 reactions)

Kras is a member of the RAS protein family, which are a class of small GTPases involved in cell signaling pathways. The Ras signaling pathway plays an important role in cell proliferation and differentiation. Conversion of Kras from the inactive GDP-bound state to the active GTP-bound state triggers the downstream effector and promotes cell growth. RAS genes are frequently mutated in various human tumors. These mutations block the GTPase activity of RAS and lock RAS in the GTP-bound state, resulting in constitutively active signals through the downstream cascades leading to cancer cell proliferation.

Assay Principle

The Kras (G12V) nucleotide exchange assay is a TR-FRET based assay. The assay kit is designed to detect the GTP binding status of wild type Kras in the presence of SOS1, the most-studied guanine nucleotide exchange factor (GEF) of Kras. The Tag2-Kras in this assay kit is recognized by a Terbium-labeled anti-Tag2 antibody (HTRF donor). If Kras binds to a fluorescence-labeled GTP (HTRF acceptor), the donor and the acceptor will be brought in close proximity. Excitation of Terbium (340 nm) generates fluorescence resonance energy transfer (FRET) to the fluorescence-labeled GTP acceptor, which consequently fluoresces at 665 nm (figure below). Thus, GTP binding to Kras can be quantitively measured by calculation of the fluorescent ratio of 665 nm/620 nm. The inhibitor blocking the nucleotide exchange will reduce the HTRF signal.