The KRAS(G12C) Nucleotide Exchange Assay is a homogeneous assay designed for the screening and profiling of KRAS(G12C) antagonists/inhibitors by using BODIPY®-GDP to monitor the GDP or GTP binding status. The KRAS(G12C) Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant KRAS(G12C) labeled with BODIPY®-GDP, GTP, assay buffer and additives for 400 enzyme reactions. The kit can be used with two different protocols for greater flexibility, either titrating the inhibitor at a fixed GTP concentration or titrating the GTP at a fixed inhibitor concentration.

BODIPY FL-GDP is a mixed isomer in which the fluorophore has been attached to the 2’ or 3’ position of the ribose ring via a linker. It is a green-fluorescent dye with similar excitation and emission to fluorescein or Alexa Fluor™ 488, characterized by a high extinction coefficient and high quantum yield and is relatively insensitive to pH changes. The dye has an excited-state lifetime of 5 nanoseconds or longer.

Assay Principle, Illustration: KRAS is activated upon binding GTP, when it 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 KRAS affinity for GTP is greater than its affinity for GDP. The fluorescence intensity decreases as the BODIPY-GDP is displaced. Several KRAS inhibitors lock KRAS in the (inactive) GDP-bound conformation and prevent GDP/GTP exchange. In this scenario the fluorescence intensity increases with drug concentration as more BODIPY--GDP remains bound to KRAS.