背景介绍 It is well established that RAS mutations are responsible more than 30% of human cancers and KRAS(G12C) is one of the KRAS mutations that is found frequently in lung and colon cancers. The G12C mutation favors the activated (GTP-bound) state of KRAS, amplifying signaling pathways that lead to oncogenesis. Recent studies discovered a small molecule called AMG510 (Amgen) that can block the KRAS(G12C)-mediated signaling pathway by locking the KRAS conformation in the GDP-bound (inactive) state. Compounds that affect the nucleotide exchange (GDP to GTP) reaction in KRAS could lead to a novel approach to the inhibition of tumor cell growth in KRAS(G12C) driven tumors. 产品介绍 The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12C) antagonists/inhibitors by monitoring the binding of an effector protein (i.e. Ras binding domain of Raf1, RBD-cRaf) to KRAS(G12C). With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP loaded KRAS(G12C) 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 is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP. GDP-loaded KRAS(G12C) is in an inactive state, and does not interact with the Ras-binding domain (RBD) of cRAF. SOS1 assists in the release GDP from KRAS(G12C) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS(G12C) that permits binding of RBD-cRAF to KRAS(G12C).
The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS (G12C) to assay binding of KRAS(G12C) to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni chelate donor beads are brought into proximal range by the binding of KRAS(G12C) and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation.