The KRAS(G12V) Isoform B Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of antagonists/inhibitors of KRAS(G12V), Isoform B, by monitoring the binding of an effector protein such as the Ras binding domain of Raf1 (RBD-cRaf) to KRAS. First, a sample containing GDP-loaded KRAS(G12V) Isoform B is incubated with SOS1 and GTP for the nucleotide exchange. Next, RBD-cRAF is added and incubated for the effector-RAS binding. Then, acceptor and donor beads are added and incubated for detection followed by reading the Alpha-counts.

SOS1 (son of sevenless) is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP. GDP-loaded KRAS(G12V) Isoform B is in an inactive state and does not interact with the Ras-binding domain (RBD) of cRAF. SOS1 assists in the release of GDP from KRAS(G12V) Isoform B so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS that permits its binding to RBD-cRAF. The KRAS (G12V) Isoform B Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS(G12V) Isoform B to assay the binding of KRAS to RBD-cRAF in the AlphaLISA® assay. Glutathione acceptor and Nickel chelate donor beads are brought into proximal range by the binding of KRAS and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation.

Figure 1: Illustration of the assay principle.