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KRAS WT/SOS1 binding kit HTRF®

The KRAS WT/SOS1 binding kit is designed to identify KRAS WT/SOS1 inhibitors.

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  • No-wash No-wash
  • All inclusive kit All inclusive kit
  • Accurate pharmacology Accurate pharmacology

The KRAS WT/SOS1 binding kit is designed to identify KRAS WT/SOS1 inhibitors.

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Overview

A fast and easy way to identify new inhibitors of KRAS WT / SOS1 protein interactions.

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 GTPase, KRAS binds to GDP in its inactive form or binds to GTP to switch into the active form. KRAS G12C is one of the most commonly represented mutant forms in cancer, which leads to a permanently active state of KRAS. The Ras guanine nucleotide exchange factor, also called SOS1, is a GEF protein promoting the active form of KRAS. The upregulation of the KRAS / SOS1 interaction leads to cancer phenotypes.



Benefits

  • CANCER RESEARCH
  • KRAS INHIBITORS

Assay principle

The KRAS WT/SOS1 PPI assay includes tagged human recombinant partners  (KRAS WT and SOS1) and labelled anti-tag reagents for HTRF detection. Without an inhibitor, KRAS WT loaded with GTP binds to SOS1, and the binding of each detection reagent to its tagged target generates an HTRF signal. In the presence of KRAS WT/SOS1 inhibitors or GTP competitors, the HTRF signal decreases.


Principle of the HTRF KRAS WT/SOS1 binding assay

Assay protocol

The Human KRAS WT/SOS1 binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, compounds or standards are dispensed directly into the assay plate, the pre-mixed GTP and human Tag1-KRAS WT protein is then added together with the human Tag2-SOS1 protein, followed by the dispensing of the HTRF reagents: the anti-Tag2 antibody labeled with Terbium cryptate and  the anti-Tag1 antibody labeled with XL665. 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.


Assay protocol of the HTRF KRAS WT/SOS1 binding assay

KRAS WT/SOS1 inhibitor screening

Various compounds known to be KRAS or SOS1 inhibitors were added to the assay. BAY-293, BI-3406, BI-2852, and MRTX-1257 display the right potency and pharmacological ranking, in good correlation with the literature. ARS-1620 and AMG-510, two KRAS G12C selective compounds, had no effect on the assay, confirming that the KRAS WT/SOS1 binding kit is specific to the KRAS WT form.

Validation of the HTRF KRAS WT/SOS1 binding kit with inhibitors

Competitive or non competitive nucleotide screening

The two nucleotides GDP and ATP were added to the assay. GDP, a GTP competitor which is well known to bind KRAS WT, displays the right potency, in good correlation with the literature. ATP, a non GTP competitive nucleotide, had no effect on the assay, confirming the specificity of the kit in detecting GTP competitors or KRAS WT / SOS1 inhibitors.

Validation with competitive or non-competitive nucleotides

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HTRF addresses large protein-protein interaction complexes

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Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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