The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS IsoformB antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS. With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS 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 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 Isoform B so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS IsoformB that permits its binding to RBD-cRAF. The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS Isoform B to assay binding of KRAS to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni 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.