The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12D) antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS(G12D). The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant GDP-loaded KRAS(G12D) Isoform A, GTP, exchange factor SOS1 (amino acids 564-1049), effector protein RBD-cRAF (amino acids 50-140), assay buffer and additives for 400 reactions. With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS(G12D) 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(G12D) 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(G12D) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS(G12D) that permits its binding to RBD-cRAF. The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS (G12D) to assay binding of KRAS(G12D) to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni chelate donor beads are brought into proximal range by the binding of KRAS(G12D) and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation.

Figure 1: Illustration of the assay principle.