DESCRIPTION

• Uniprot: P01116
• Shiptemp: -80°C (dry ice)
• Warnings: Avoid freeze/thaw cycles
• Category: Cell Signaling/Assay Kit
• Background: High levels of wild-type KRAS cause a slowing of cell replication and growth, and increased apoptosis. This can be induced by cellular stress, certain types of radiation, chemical signals, and other prompts. Wild-type KRAS may also protect against mutant KRAS over-activation by dimerizing with mutant KRAS protein. The study of differences in the behavior of the wild-type and mutated forms of KRAS is especially important since KRAS mutations are responsible for more than 30% of human cancers. Compounds that affect the nucleotide exchange (GDP to GTP) reaction provide a novel approach to the inhibition of tumor cell growth in KRAS-driven tumors. In 2021, the Food and Drug Administration granted approval for use of AMG510 (Sotorasib), a potent KRAS G12C inhibitor for non-squamous non-small cell lung cancer.
• Description: The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS IsoformB antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS. With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS Isoform B is incubated with SOS1 and GTP for the nucleotide exchange. Next, RBD-cRAF is added and incubated for the effector-RAS binding. Then, acceptor and donor beads are added and incubated for detection followed by reading the Alpha-counts. SOS1(son of sevenless)is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP. GDP-loaded KRAS Isoform B is in an inactive state and does not interact with the Ras-binding domain (RBD) of cRAF. SOS1 assists in the release of GDP from KRAS Isoform B so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS IsoformB that permits its binding to RBD-cRAF. The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS Isoform B to assay binding of KRAS to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni chelate donor beads are brought into proximal range by the binding of KRAS and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation. Figure 1: Illustration of the assay principle. 
• Methodology: AlphaLISA®
• Supplied As: The KRAS IsoformB Coupled Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant GDP-loaded KRAS Isoform B, GTP, exchange factor SOS1, an effector protein RBD-cRAF, assay buffer and additives for 400 reactions.
• Unspsc Code: 41105332
• Unspsc Name: Protein chemiluminescent detection reagents or kits or substrates
• Applications: • Screen small molecule inhibitors or antagonists that affect KRAS Isoform B nucleotide-binding status in high throughput (HTS) applications. • Counter-screen for the compounds that affect mutated forms of KRAS
• Product Type: Assay Kit
• Biosafety Level: Not applicable (BSL-1)
• Related Products: 79859, 78355, 78519, 78565
• Contraindications: Green and blue dyes, such as Trypan Blue, absorb light in the AlphaScreen® signal emission range (520-620 nm). Avoid the use of the potent singlet oxygen quenchers such as sodium azide (NaN3) or metal ions (Fe2+, Fe3+, Cu2+, Zn2+ and Ni2+). The presence of >1% RPMI 1640 culture medium leads to a signal reduction due to the presence of excess biotin and iron in this medium. MEM, which lacks these components, does not affect AlphaScreen® assays.
• Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
• Scientific Category: Cell Signaling Pathway