Kras Related Assays
Target-specific Assays

RAS Assays for Drug Discovery

RAS is a small guanosine triphosphate (GTP)-binding protein (GTPase) that is frequently mutated in a large percentage of cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful.

The recent identification of a unique binding pocket and successful inhibition of the KRAS G12C mutant by covalent chemical modifiers have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Alternative efforts are undertaken for the inhibition of interactions of KRAS with exchange factors and effector proteins.

Reaction Biology is committed to quality service in every aspect of our relationships with clients: before, during, and after the study, the assigned business development manager will be just one phone call away to answer any questions. Robust and reproducible assay formats for our RAS assay suite ensure high-quality data.

RAS-targeted Therapies

Download our poster, authored by Frank McCormick et al. and published by Nature Reviews Drug Discovery, that summarizes the key technological advancements and drug discovery projects targeting the RAS family of proteins.

Download our poster here.

RAS Nature Poster

RAS-related Assays List

* Please inquire

NEA … Nucleotide Exchange Assay
PPI  … Protein:Protein Interaction
SPR … Surface Plasmon Resonance
TSA … Thermal Shift Assay

Biochemical and Biophysical KRAS Assays

  • RAS Protein:Protein Interaction Assays
  • RAS Nucleotide Exchange Assay
  • KRAS Mutant NEA Panel
  • Thermal Shift Direct Binding Assay
  • Surface Plasmon Resonance Direct RAS Binding Assay
RAS Protein:Protein Interaction Assays

RAS::SOS1 Protein:Protein Interaction (PPI) Assay

Disruption of SOS1 binding to KRAS can be used as an orthogonal method for studying SOS1 specific compounds. The assay uses an HTRF-based detection of interaction.


cRAF recognizes the GTP-bound form of RAS. cRAF binding assay can be used for the identification of disruptors of interaction between Ras and cRAF, as well as quantification of the nucleotide exchange reaction. This assay can be used as an alternative to the regular Nucleotide Exchange Assay with an optional examination of SOS1 independent GTP binding. The assay uses an HTRF-based detection of interaction.

Please inquire about custom-tailored GTPase assay development.

The PPI assay is available for wild-type RAS and various RAS G12 mutants.


RAS Nucleotide Exchange Assay

The RAS NEA allows the monitoring of SOS1/2 mediated exchange of fluorescently labeled GDP to GTP.

The main application of the assay is to identify compounds that lock RAS in the inactive “OFF” state by preventing GTP binding.

An alternative NEA format utilizes GTP labelled with DY-647P1 and monitors the increase in HTRF signal observed upon GTP binding to RAS. The assay is performed at lower GTP concentrations compared to the standard RAS NEA and can evaluate various modes of nucleotide exchange inhibition.

Please inquire about custom-tailored assay development.

The RAS NEA is available for wild-type RAS and various G12 mutants thereof.

KRAS Mutant NEA Panel

The KRAS mutants NEA (Nucleotide Exchange Assay) selectivity panel is designed for screening and profiling of potent inhibitors against KRAS mutants.

The HTRF (Homogeneous Time Resolved Fluorescence) based NEA assay employs GTP labeled with DY-647P1 and monitors the increase in HTRF signal observed upon GTP binding to RAS. The assay can evaluate various modes of nucleotide exchange inhibition.

The primary goal of the panel is to help scientists evaluate compound specificity between various KRAS mutants and the other two RAS homologs.

The following proteins are currently included in the panel:


The panel will run on a monthly basis.
Please inquire about custom-tailored assay development.

A schematic showing the specificity of two compounds, MRTX-849 and BI-2852, against various KRAS mutants and two RAS homologs.

Thermal Shift Direct Binding Assay

Thermal Shift Assays (TSAs) are used to assess the effects of compounds on protein stability. Selectivity of two compounds KRAS mutant G12C is shown.

The TSA is available for RAS and various G12 mutants thereof as well as SOS1 and SOS2. Please inquire about custom-tailored assay development.

Surface Plasmon Resonance Direct RAS Binding Assay

Surface Plasmon Resonance (SPR) is used to quantify the binding affinity of the molecule as well as binding kinetics. A comparison between KRAS WT and mutant proteins can be performed to determine selectivity.

KRAS and various G12 mutants thereof as well as SOS1 are established for SPR analysis. Please inquire for custom-tailored assay development.

Example study: The KRAS G12D mutant selective peptide KRpep-2d was used to show the difference in the binding of KRpep-2d to mutant G12D versus wild type KRAS and other mutants. The peptide binds to all targets, however, the binding affinity (KD) of the peptide is 15 times higher when interacting with the G12D mutant.

RAS Binding Assays via NanoBRET Technology

  • NanoBRET Technology
  • Assay Principle
NanoBRET Technology

Testing compound binding to the target in the physiologic environment of intact cells is the ideal assay to bridge from biochemical to phenotypic compound testing in cellular tumor models.

Advantages of the NanoBRET Target Engagement RAS Assay:

  • Testing of compound-target binding in intact cells
  • Determination of binding affinity and target protein occupancy as well as residence time in the intracellular environment
  • A variety of orthosteric and allosteric inhibitors to RAS can be tested
  • Multiwell assay suitable for medium-throughput upscaling
  • High reproducibility
Assay Principle

The NanoBRET assay employs an energy transfer technique designed to measure molecular proximity in living cells. The assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET tracer, reversibly bound to a NanoLuc luciferase-RAS fusion construct in cells.

The NanoLuc luciferase is a split NanoLuc construct, consisting of a large luciferase part fused to RAS and a small luciferase part fused to Ras that are both expressed in the cells. Upon oligomerization of RAS molecules, the small and large luciferase parts constitute one functional luciferase molecule. Please view the Promega webinar on NanoBRET RAS assays for more information here starting at 17:10 minutes:seconds.

The intracellular binding affinity and selectivity are physiologically relevant and fundamental to the pharmacological mechanism of the compounds. While biochemical and biophysical assays identify the RAS inhibitors in vitro, the NanoBRET assay serves as a great tool to determine the direct interaction of the compounds binding to RAS in cells.

The assay is available for KRAS and HRAS as well as mutants thereof. Please refer to the table above for datasheets featuring example data.

KRAS 3D Spheroid and Cellular Phosphorylation Activity Assays

  • Assay Technology
Assay Technology

KRAS Inhibitor Activity Screening using 3D Spheroid Cellular Phosphorylation Assay

The MAPK signaling cascade is regulated by KRAS signaling leading to changes in the activation status of MEK1 and subsequent phosphorylation of downstream targets such as ERK1/2 (pT202/pY204) (Figure 1). It has been shown that missense single-base mutations such as G12C or G12D result in constitutive activation due to impaired GTP hydrolysis1.

Our Cellular Phosphorylation service allows for the study of the direct effects of KRAS inhibitors against different KRAS mutants.  The assay can help determine the activity of KRAS inhibitors in a live cell environment. Since inhibitors of that pathway have been shown to more efficiently kill cells growing as 3D organoids instead of in 2D,  we have moreover established 3D-spheroid assays for the analysis of phenotypic effects of KRAS inhibitors. View our recent poster presented at AACR 2023 which demonstrates the use of the cellular phosphorylation and 3D spheroid assays to evaluate the activity and selectivity of KRAS inhibitors.

Cellular ERK(pT202/pY204)-phosphorylation Assays are available and validated for the following cell lines:

Target Cell Line KRAS Mutation Status
MEK1/2 PANC-1 G12D
Mia-Pa-Ca-2 G12C
NCI-H358 G12C
SW-620 G12V
HT-29 WT

All of these cell lines are also established in the 3D spheroid assay.

To enquire about additional cell lines or request more information: Contact us


Figure 1: Figure showing how tyrosine kinase receptor signaling is modulated by KRAS. One can interrogate various KRAS mutants using cell lines that express specific KRAS mutations2.


1. Kessler, D. et al. Proc Natl Acad Sci U S A 116, 15823–15829 (2019).
2. Zhu, G. et al. Mol Cancer 20, 143 (2021).

RAS-related Recombinant Proteins

  • RAS and RAS mutants
  • RAS-pathway proteins
RAS and RAS mutants

KRAS, NRAS, and HRAS recombinant proteins are available with a variety of tags.

Available mutants: G12C, G12D, G12R, G12V, Q61H, G12D-T35S. G13 mutant variants are in production.

Our RAS-related recombinant proteins are available for purchase, please visit our products page or inquire for more information.

RAS-pathway proteins

A variety of RAS pathway related recombinant proteins were produced in house and are available for screening.

Please see a complete list in our product shop.

Cell Line-derived Models for In Vitro and In Vivo Testing of RAS Inhibitors

Drug testing on tumor cells that carry RAS mutations is performed with the goal to elucidate phenotypic effects of treatment and determine the potency of new drug candidates. Reaction Biology provides testing on conventional secondary cell culture and cell-line derived xenograft models.

Overall, RAS inhibitors show a higher potency in 3D models than in 2D models, as exemplified in our poster Characterization of KRAS Inhibitors in 2D and 3D Cellular Assays.

Our in vivo models are suited to study the effect of cancer drugs on tumors derived from cell lines. Our cell line-derived xenograft models are available with a variety of tumor placement options including subcutaneous, orthotopic, and metastasis models.