KRAS(G12C) Coupled Nucleotide Exchange Assay Kit (Discontinued)

Catalog #

78004

$1,490 *

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Description

The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit is designed for screening and profiling of KRAS(G12C) antagonists/inhibitors by monitoring the binding of an effector protein (i.e. Ras binding domain of Raf1, RBD-cRaf) to KRAS(G12C). The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant GDP-loaded KRAS(G12C) Isoform A, GTP, exchange factor SOS1, an effector protein RBD-cRAF, assay buffer and additives for 400 reactions. With this kit, a few simple steps on a microtiter plate are required for nucleotide exchange detection. First, a sample containing GDP-loaded KRAS(G12C) 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 is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP. GDP-loaded KRAS(G12C) is in an inactive state, and does not interact with the Ras-binding domain (RBD) of cRAF. SOS1 assists in the release GDP from KRAS(G12C) so that GTP can occupy the nucleotide binding pocket. This results in a conformational change in KRAS(G12C) that permits binding of RBD-cRAF to KRAS(G12C). The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit utilizes GST-tagged RBD-cRAF and His-tagged KRAS (G12C) to assay binding of KRAS(G12C) to RBD-cRAF in the Alpha assay. Glutathione acceptor and Ni chelate donor beads are brought into proximal range by the binding of KRAS(G12C) and RBD-cRAF, enabling the energy transfer from the donor to acceptor beads after laser excitation.

SOS1 is a guanine nucleotide exchange factor that facilitates the exchange of GDP for GTP

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Synonyms

C-K-RAS kit, CFC2 kit, K-RAS2A kit, Coupled Nucleotide Exchange Assay, SOS1 kit

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Product Data Gallery

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Supplied As

The KRAS(G12C) Coupled Nucleotide Exchange Assay Kit comes in a convenient 384-well format, with enough purified recombinant GDP-loaded KRAS(G12C) Isoform A, GTP, exchange factor SOS1, an effector protein RBD-cRAF, assay buffer and additives for 400 reactions

Materials Required But Not Supplied

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

Format

Catalog Number	Name	Amount	Storage
100640	GDP loaded KRAS(G12C), Isoform A, His-tag*	10 μg	-80°C
100753	SOS1, FLAG-Tag, Avi-Tag, Biotin-Labeled*	2 x 100 µg	-80°C
100519	RBD-cRAF, GST-tag	5 µg	-80°C
79861-2	GTP (10 mM)	0.5 ml	-20°C
78351	RBD-RAS Binding Buffer	6 ml	4°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.

UniProt #

P01116

Background

It is well established that RAS mutations are responsible for more than 30% of human cancers. KRAS(G12C) is one of the KRAS mutations that is found frequently in lung and colon cancers. The G12C 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 AMG510 (Amgen) that locks KRAS conformation in the GDP-bound (inactive) state, thereby blocking KRAS(G12C)-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(G12C) driven tumors.

References

1. Canon, J., et al. Nature 2019; 575:217-223.
2. Hillig, R.C., et al. 2019. PNAS USA 116 (7): 2551-2560

Storage/Stability

At least one year from date of receipt when stored as directed.

Instructions for Use

See assay protocol for deatiled instructions.

Applications

1. Screen small molecule inhibitors or antagonists that are expected to affect KRAS(G12C) nucleotide-binding status for drug discovery and high throughput (HTS) applications.

Shipping Temperature

-80°C

Notes

Troubleshooting Guide
Visit bpsbioscience.com/assay-kits-faq for detailed troubleshooting instructions. For all further questions, please email [email protected]

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.

The final concentration of DMSO in the reaction should not exceed 1%.

Disclaimers and Limitations

BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.

Scientific Category

Ras Signaling

Regulatory Status

Research Use Only

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KRAS(G12C) Coupled Nucleotide Exchange Assay Kit (Discontinued)

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