KRAS(G12D) Nucleotide Exchange Assay Kit
The KRAS(G12D) Nucleotide Exchange Assay is a homogeneous assay designed for the screening and profiling of KRAS(G12D) antagonists/inhibitors by using BODIPY®-GDP to monitor the GDP or GTP binding status. 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 BODIPY® FL 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® FL 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. KRAS inhibitor MRTX-1133 locks KRAS in the (inactive) GDP-bound conformation and prevents GDP/GTP exchange. In this scenario the fluorescence intensity increases with drug concentration as more BODIPY GDP remains bound to KRAS.
- Fluorescent microplate reader capable of reading λex/em=470 nm/525 nm
- Adjustable micropipettor and sterile tips
Catalog # | Name | Amount | Storage |
100887 | KRAS (G12D), Isoform A, His-Tag, BODIPY®-GDP Loaded, 5 µM | 60 µg x 4 | -80°C |
79861-1 | Guanosine 5′-triphosphate (GTP), 10 mM | 100 µl | -20°C |
79862 | 2X KRAS Nucleotide Exchange Buffer | 5 ml | -20°C |
DTT, 0.5 M | 200 µl | -20°C | |
EDTA, 0.5 M | 100 µl | Room Temp | |
79961 | 384-well plate, Black | 1 | Room Temp |
Plate sealing film | 1 |
Note: The molecular weight of the protein is 23 kDa. The volume provided is ≥520 µl/tube (x4 tubes), which is sufficient for 5 µl/well as described in the protocol
It is well established that RAS mutations are responsible for more than 30% of human cancers. KRAS(G12D) is one of the KRAS mutations that is found frequently in pancreatic and colon cancers. Recent studies have led to the discovery of a small molecule called MRTX-1133 that locks KRAS conformation in the inactive GDP-bound state, thereby blocking the KRAS(G12D)-mediated signaling pathway. Compounds that affect the nucleotide exchange (GDP to GTP) reaction in KRAS may inhibit tumor cell growth in KRAS(G12D)-driven tumors.