KRAS(G12C) Nucleotide Exchange Assay Kit
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 preloaded 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.
- Fluorescent microplate reader capable of reading λex/em=470 nm/525 nm
- Adjustable micropipettor and sterile tips
| Catalog # | Name | Amount | Storage |
| 100537 | KRAS (G12C), 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 (Assay 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 |
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(G12C) is the most frequent mutation, found in lung and colon cancers. Recent studies uncovered a small molecule called AMG510 (Amgen) that inhibits the KRAS(G12C)-mediated signaling pathway by locking the KRAS conformation in the GDP-bound state. Compounds that block the nucleotide exchange (GDP to GTP) reaction in KRAS could lead to the inhibition of tumor cell proliferation in KRAS(G12C)-driven tumors.
Ostrem, J. M., et al., Nature. 2013; 503:548-551.
Patricelli, M. P., et al., Cancer Discovery. 2016; 6:316-329
Powered by Bioz
San Diego, CA 92121
858-202-1401
Products are for Research Use Only
COMPANY
Copyright © 2024
BPS Bioscience, Inc.
All rights reserved.
Diagrams on this website are created with BioRender.com.