Product Description

KRAS[G12D] is one of the most common activating mutations of KRAS, causing defective GTP hydrolysis and constitutive KRAS activation. RAF1 (also known as C‑RAF) is a serine/threonine kinase that acts as a key downstream effector of RAS; upon binding to GTP‑bound KRAS[G12D], RAF1 is activated and subsequently phosphorylates MEK1/2, initiating the MAPK cascade that regulates cell proliferation, differentiation, and survival. Under normal conditions, KRAS‑RAF1 signaling is tightly regulated; however, in KRAS[G12D]‑mutant tumors, this signaling is constitutively active, driving tumor growth and drug resistance. KRAS[G12D] mutations are prevalent in various malignancies, including pancreatic, colorectal, and non‑small cell lung cancers. Inhibitors targeting the KRAS[G12D]‑RAF1 interaction (e.g., direct mutant RAS binders or RAF inhibitors) are under active development, and although RAF inhibitors may be limited by feedback activation, combination strategies or novel mutant‑selective inhibitors (such as KRAS G12D covalent inhibitors) hold promise to overcome these challenges, underscoring the critical value of this interaction in cancer therapy.

Screeningbio’s TR-FRET GDP‑loaded KRAS[G12D]/RAF1 Binding Assay Kit can be used to measure the interaction between KRAS[G12D] (loaded with GDP) and RAF1 protein. This kit, developed using TR‑FRET technology, is suitable for high‑throughput screening of small‑molecule inhibitors, peptides, or antibody blockers, and is characterized by simplicity, rapidity, and high accuracy.

The basic principle of this method is as follows: an anti‑Tag 2 antibody conjugated to Solar Eu*1 specifically recognizes RAF1 protein bearing Tag 2, while an anti‑Tag 1 antibody conjugated to LA*2 recognizes KRAS[G12D] protein bearing Tag 1. When RAF1 binds to KRAS[G12D], the tag antibodies conjugated to Solar Eu and LA, respectively, are brought into close proximity. Upon excitation by an external light source, fluorescence resonance energy transfer occurs between the donor and acceptor. The interaction level between KRAS[G12D] and RAF1 protein can be determined by measuring the signal intensity at a specific wavelength (665 nm). Consequently, compounds, peptides, or antibodies that block the KRAS[G12D]/RAF1 interaction will result in a decrease in the TR‑FRET signal.