Kras (G12R) -cRAF Binding Assay Kit

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 regulates diverse cellular processes, including cell proliferation, differentiation, and survival. Conversion of Ras from the inactive GDP-bound state to the active GTP-bound state activates the downstream effector and promotes cell growth. RAF is a key downstream effector of RAS. Since the frequently mutated Ras genes are associated with various human tumors, the Ras-RAF signaling pathway is considered a potential therapeutic target for cancer treatment.

Assay Principle

The Kras (G12R)-cRAF binding assay kit is a TR-FRET based assay, which is designed to detect the binding status between Kras and cRAF. Tag2-Kras (G12R) in this assay kit is loaded with GppNHp, which represents the activated Kras. The Ras binding domain (RBD) of cRAF has a Tag1 at N-terminus. A Terbium-labeled anti-Tag2 antibody binding to the Tag2-Kras serves as a fluorescence donor (HTRF donor), activation of which results in fluorescence resonance energy transfer (FRET) if the Tag1-cRAF binds to Kras, since the binding brings Terbium on the anti-Tag2 antibody close to the fluorophore on the anti-Tag1 antibody (HTRF acceptor). Thus, the binding status can be quantitively measured by calculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). Blocking the Kras-cRAF binding will reduce the HTRF signal.