KRAS WT GTP binding kit HTRF®

Associated products

The KRAS WT GTP binding kit is designed to identify KRAS WT / GTP inhibitors.

No-wash
All inclusive kit
Accurate pharmacology

Download the protocols

The KRAS WT GTP binding kit is designed to identify KRAS WT / GTP inhibitors.

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Overview

A fast and easy way to identify new KRAS WT / GTP competitors.

KRAS is a small GTPase implicated in various biological processes, such as cell proliferation, cell survival, and cell metabolism. This proto-oncogene is well known to be mutated in many cancer subtypes, inducing an uncontrolled proliferation and cell metabolism modifications. It thereby contributes to the Warburg effect in cancer cells. Like the majority of small GTPases, KRAS binds to GDP in its inactive form or binds to GTP to switch to the active form. KRAS G12C is one of the most commonly present mutant forms in cancer which lead to a permanently active state of KRAS. Identifying new KRAS / GTP competitors is therefore a relevant strategy to control biological processes involved in cancer growth by reducing the KRAS activity, as well as the associated pathways.

Benefits

CANCER RESEARCH
KRAS INHIBITORS

Assay principle

The HTRF KRAS GTP binding assay is in a competitive assay format, using a GTP-Red reagent as KRAS WT ligand, a 6His tagged human KRAS WT protein, and an anti 6His Cryptate-labeled antibody. The compound being tested competes with the GTP Red reagent, and thereby prevents FRET from occurring.

Principle of the HTRF KRAS WT GTP competitive assay

Assay protocol

The Human KRAS WT binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate. The human His-tagged KRAS WT protein is then added, followed by the dispensing of the HTRF reagents: The anti 6His antibody labeled with Europium cryptate and the GTP-Red reagent. The reagents labeled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

GTP-Red reagent Kd	70nM
GTP-Red reagent concentration	50nM
KRAS GTP binding Standard (GDP) IC50	35nM
KRAS GTP binding Standard Ki	20.4nM

Screening of GTP competitors with human KRAS WT protein

Various nucleotides known to be GTP competitors were added to the assay. GDP (assay standard), GTPyS, and GTP displayed the right potency, in good correlation with the literature. ATP, a non-specific nucleotide, and BAY-293, a KRAS/SOS1 inhibitor, had no effect on the assay, confirming the specificity of the kit.

Validation of the HTRF KRAS WT GTP binding kit with nucleotides

Validation with competitive or non-competitive compounds

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Deciphering low- and high affinity interactions - Application Notes

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Monitoring nuclear receptor binding with HTRF assays - Application Notes

HTRF addresses large protein-protein interaction complexes

Challenge large complexes with HTRF assays - Application Notes

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Product Insert KRAS WT GTP Kit / 64BDKRASWPEG-64BDKRASWPEH

64BDKRASWPEG-64BDKRASWPEH - Product Insert

KRAS GTP competitors and KRAS/SOS1 inhibitors screening with new HTRF biochemical-based assays

Screening for KRAS competitors and inhibitors with HTRF technology - Application Notes

Safety Data Sheet (DEU) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (ELL) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (FRA-FR) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (ITA) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (SPA) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (ENG-GB) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (ENG-US) KRAS WT GTP Kit / 64BDKRASWPEG

64BDKRASWPEG - Safety Data Sheet

Safety Data Sheet (DEU) KRAS WT GTP Kit / 64BDKRASWPEH