KRAS (G12V) Nucleotide Exchange Assay Kit

The KRAS (G12V) Nucleotide Exchange Assay Kit is a fluorogenic homogeneous assay designed for the screening and profiling of KRAS (G12V) antagonists/inhibitors by using BODIPY®-GDP to monitor the KRAS (G12V) GDP or GTP binding status. The KRAS (G12V) Nucleotide Exchange Assay Kit comes in a convenient format, with enough purified recombinant KRAS (G12V) isoform B 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 theKRAS (G12V) 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.

Product Specifications

Background

It is well established that RAS mutations are responsible for more than 30% of human cancers. Although new treatments have been recently developed to mitigate the tumor-promoting effects of RAS mutations, one mutant, KRAS (G12V), has been resistant to most known inhibitors. For example, the small molecule AMG510 (Amgen) can block the signaling pathway mediated by several KRAS (G12) mutants, by locking the KRAS conformation in the GDP-bound state. However, KRAS (G12V) has been insensitive to this inhibitor. New compounds that affect the nucleotide exchange (GDP to GTP) reaction in KRAS mutant G12V are expected to inhibit tumor cell growth in KRAS (G12V) -driven tumors. MRTX1133, which selectively inhibits the KRAS G12D mutant but not KRAS wild-type, has shown promising activity on KRAS (G12V) in vitro. The development of new highly specific inhibitors will open therapeutic avenues.

UniProt

P01116

Applications

Study enzyme kinetics and screen small molecular inhibitors of KRAS (G12V) for drug discovery and High Throughput (HTS) applications.

Format

Catalog #NameAmountStorage101504KRAS (G12V), Isoform B, BODIPY®-GDP Loaded, His-Tag*2 x 60 µg-80°C79861-1Guanosine 5′-triphosphate (GTP) (10 mM) 100 µl-20°C798622X KRAS Nucleotide Exchange Buffer (Assay Buffer) 5 ml-20°C827350.5 M DTT200 µl-20°C827630.5 M EDTA100 µlRoom Temp7968596-well black microplate1Room Temp*The molecular weight of the protein is 23 kDa. The volume provided is ≥520 µl/tube (x2 tubes), which is sufficient for 10 µl/well as described in the protocol.Catalog #NameAmountStorage101504KRAS (G12V), Isoform B, BODIPY®-GDP Loaded, His-Tag*4 x 60 µg-80°C79861-1Guanosine 5′-triphosphate (GTP) (10 mM) 100 µl-20°C798622X KRAS Nucleotide Exchange Buffer (Assay Buffer) 2 x 5 ml-20°C827350.5 M DTT200 µl-20°C827630.5 M EDTA100 µlRoom Temp79961384-well black microplate1Room Temp*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.

Shipping Conditions

-80°C

Storage Conditions

This assay kit will perform optimally for up to6 monthsfrom date of receipt when the materials are stored as directed.

Notes

Troubleshooting GuideVisitbpsbioscience.com/assay-kits-faqfor detailed troubleshooting instructions. For all further questions, please email[email protected].