KRAS(G12D) Coupled Nucleotide Exchange Assay Kit

Catalog #

78570

$1,355 *

Size: 384 reactions

Qty

Bulk/Custom

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*US Pricing only. For international pricing, please contact your local distributor.

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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). 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 (amino acids 564-1049), effector protein RBD-cRAF (amino acids 50-140), 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(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.

KRAS(G12D) Coupled Nucleotide Exchange Assay Kit

Figure 1: Illustration of the assay principle.

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Product Data Gallery

Nucleotide exchange of KRAS (G12D).

Effect of MRTX1133 and AMG510 on the nucleotide exchange of KRAS(G12D).

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Materials Required But Not Supplied

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

Format

Catalog #	Name	Amount	Storage
101312	KRAS(G12D), Isoform A, His-Tag, GDP-Loaded *	5 µg	-80°C
101573	SOS1, FLAG-Tag*	50 µg	-80°C
100519	RBD-cRAF, GST-Tag*	5 µg	-80°C
79861-2	10 mM GTP	0.5 ml	-20°C
	RBD-RAS Binding Buffer (Incomplete)	2 x 3 ml	-20°C
	0.5 M DTT	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.

UniProt #

P01116

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.

References

Wang, X., et al., 2022 J Med Chem 65: 3123-33.
Hillig, R.C., et al., 2019 PNAS USA 116 (7): 2551-2560

Storage/Stability

This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed. Avoid multiple freeze/ thaw cycles!

Applications

Screen small molecule inhibitors or antagonists that affect KRAS(G12D) nucleotide-binding status in high throughput screening (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 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%.

Warnings

Avoid freeze/thaw cycles

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.

End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.

Scientific Category

Ras Signaling

Regulatory Status

Research Use Only