KRAS (G12D) Nucleotide Exchange Assay Kit

The KRAS (G12D) Nucleotide Exchange Assay Kit is a fluorogenic homogeneous assay designed for the screening and profiling of KRAS (G12D) antagonists/inhibitors by using BODIPY®-GDP to monitor the KRAS (G12D) GDP or GTP binding status. The KRAS (G12D) Nucleotide Exchange Assay Kit comes in a convenient format, with enough purified recombinant KRAS (G12D) isoform A labeled with BODIPY®-GDP, GTP, assay buffer and additives. Two different protocols can be used with this kit, allowing greater experimental flexibility. It can used to either titrate the inhibitor at a fixed GTP concentration, or to titrate GTP in the presence of a fixed inhibitor concentration.Figure 1: Schematic representation of the mechanism of the KRAS (G12D) Nucleotide Exchange Assay Kit.KRAS is activated upon binding GTP and undergoes a conformational change. KRAS then returns to a GDP-bound inactive state following the hydrolysis of GTP to GDP. In this assay, KRAS is pre-loaded with fluorescent BODIPY-GDP and therefore is inactive. Adding GTP in the presence of EDTA displaces BODIPY-GDP because the affinity of KRAS for GTP is greater than its affinity for GDP. The fluorescence intensity decreases as the BODIPY-GDP is displaced, since free BODIPY-GPD has low fluorescence versus when protein-bound. Several KRAS inhibitors lock KRAS in the (inactive) GDP-bound conformation and prevent GDP/GTP exchange. In this scenario the fluorescence intensity remains high and corelates with drug concentration as more BODIPY--GDP stays bound to KRAS.