Description
Overview
A fast and easy way to identify new KRAS G12C/GTP competitors.KRAS is a small GTPase implicated ous 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 into active form. KRAS G12C is one of the most commonly present mutant forms in cancer which leads to a permanently active state of KRAS. ying new KRAS/ GTP competitors efore a relevant strategy rol biological processes involved in cancer growth, by reducing the KRAS activity as well as the associated pathways.
How it works
Assay principle
The HTRF KRAS GTP binding assay is in a competitive assay format which uses a GTP-Red reagent as KRAS G12C ligand, a 6His tagged human KRAS G12C protein, and an anti 6His Cryptate-labeled antibody. The compound being tested competes with the GTP Red reagent, and thereby prevents FRET from occurring.
Assay protocol
The Human KRAS G12C 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 G12C protein is then adde,d followed by the dispensing of the HTRF reagents: The anti 6His antibody labeled with Europium cryptate and the GTP-Red reagent. The reagents labelled 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.
