KRAS Isoform B Coupled Nucleotide Exchange Assay Kit
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.
Name | Ordering Information |
AlphaLISA® Glutathione acceptor beads, 5 mg/ml | PerkinElmer #AL109C |
AlphaScreen® Nickel Chelate donor beads, 5 mg/ml | PerkinElmer #AS101D |
Optiplate - 384 | PerkinElmer #6007290 |
AlphaScreen® microplate reader | |
Adjustable micropipettor and sterile tips |
Catalog # | Name | Amount | Storage |
101522 | GDP-loaded KRAS Isofrom B,His-Tag* | 5 µg | -80°C |
101573 | SOS1, FLAG-Tag* | 50 µg | -80°C |
100519 | RBD-cRAF, GST-Tag* | 5 µg | -80°C |
79861-2 | GTP (10 mM) | 0.5 ml | -20°C |
RBD-RAS Binding Buffer(Incomplete) | 2 x 3 ml | -20°C | |
DTT (0.5 M) | 2 x 200 µl | -20°C | |
79311 | 3x Immuno Buffer 1 | 4 ml | -20°C |
*The concentration of the protein is lot-specific and will be indicated on the tube
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.