DESCRIPTION

• Mutation: G12D
• Shiptemp: -80°C (dry ice)
• Warnings: Avoid freeze/thaw cycles.
• Category: Ras Signaling
• Background: It is well established that RAS mutations are responsible for more than 30% of human cancers. KRAS(G12D) is the most common mutation (33%) among KRAS mutant tumors. The G12D mutation favors the activated (GTP-bound) state of KRAS, amplifying signaling pathways that lead to oncogenesis. Recent studies have led to the discovery of a small molecule called MRTX1133 (Mirati) that locks KRAS conformation in the GDP-bound inactive state, thereby blocking KRAS(G12D)-mediated signaling pathway.  Compounds that affect the nucleotide exchange (GDP to GTP) reaction could lead to a novel approach leading to the inhibition of tumor cell growth in KRAS(G12D) driven tumors.
• Description: The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12D) antagonists/inhibitors by monitoring the binding of an effector protein such as the Ras binding domain of Raf1, (RBD-cRaf) to KRAS(G12D). With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS(G12D) 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(G12D) 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(G12D) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS(G12D) that permits its binding to RBD-cRAF. The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS (G12D) to assay binding of KRAS(G12D) to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni chelate donor beads are brought into proximal range by the binding of KRAS(G12D) and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation. 
• Methodology: AlphaLISA®
• Supplied As: The KRAS(G12D) Coupled Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant GDP-loaded KRAS(G12D) Isoform A, GTP, exchange factor SOS1, 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(G12D) nucleotide-binding status in high throughput (HTS) applications.
• Product Type: Assay Kit
• Biosafety Level: Not applicable (BSL-1)
• Related Products: 79859, 100623, 78355, 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._x000D_ The final concentration of DMSO in the reaction should not exceed 1%.
• 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: Ras Signaling