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RAS is the most frequently mutated oncogene in human cancers, and KRAS is the most frequently mutated subtype of the RAS family.  Most lung, pancreatic and colorectal cancers are driven by KRAS mutations.  Until the recent discovery of KRAS (G12C)-specific covalent inhibitors, KRAS was considered an undruggable target.

Promega offers several assays and technologies to facilitate RAS drug discovery. These assays measure the binding of compounds to pathway components and assess their impact on protein:protein interactions, protein levels and, ultimately, activation of ERK.

Interested in more information about RAS pathway drug discovery? We can customize a virtual presentation to answer your questions.

Request a Seminar

Active, GTP-bound Ras activates RAF, followed by phosphorylation and activation of MEK, which is followed by phosphorylation and activation of ERK.

  • RAS: A family of small GTPases that includes KRAS, HRAS and NRAS
  • RAF: A serine/threonine kinase family consisting of c-Raf, B-Raf and A-Raf
  • MEK: A mitogen activated protein kinase kinase (MAPK/ERK Kinase)
  • ERK: An extracellular signal-regulated kinase family, also known as a mitogen activated protein kinase (MAPK), consisting of ERK1 and ERK2 that share ~85% sequence identity
Ras RAF MEK ERK pathway

An overview of the RAS-RAF-MEK-ERK pathway.

Contact us to learn more about our RAS pathway profiling and assay development services.

Target Engagement

NanoBRET™ technology offers a sensitive, specific method to measure the interaction of small-molecule drugs with their target protein in live cells.

With the NanoBRET™ Target Engagement Assay, you can:

  • Quantitate compound affinity (how tightly it binds to a protein) and target protein occupancy (how much compound binds to a protein) in live cells.
  • Assess how long a compound binds to the target protein (its residence time) under physiological conditions.
  • Scale the simple, multiwell assay to suit your research throughput needs.
  • Generate high‐quality data with low error rates and high reproducibility.
  • Get started quickly with available ready-to-use assays.
NanoBRET target engagement assay measures the interaction of small-molecule drugs with their target proteins s

Principle of the NanoBRET™ Target Engagement Assay.

RAS ERK target engagement assay data

ERK2 target engagement assay. The indicated compounds were titrated to measure occupancy with a ERK2 fusion protein in live cells.

For a complete list of Kinase Target Engagement Assays including RAF, MEK and ERK, see our Selection Table.

New PAN RAS Target Engagement Assay

We have developed a pan-KRAS NanoBRET™ tracer that has the capacity to detect a variety of orthosteric and allosteric engagement mechanisms at this challenging target, previously deemed “undruggable”.


Pan-kras assay data


pan-kras assay

Pan-KRAS NanoBRET™ target engagement assay. IC50 values were determined for the test compounds in live cells (Panel A) and cell lysates (Panel B).

Contact us for more information on our pan-KRAS assays.

Protein Degradation with HiBiT Tagging

By using CRISPR/Cas9 gene editing to knock in a bioluminescent tag at endogenous loci of interest, proteins expressed by the native promoter can be studied using simple light output measurements. Compared to overexpression models, endogenously tagged proteins maintain natural epigenetic regulation and stoichiometry with native interacting partners, resulting in improved assay windows, minimized artifacts, and more accurate models of protein biology under physiologically relevant conditions. 

HiBiT protein degradation overview

An illustration of HiBiT:LgBiT complementation to study protein degradation within a cell.

Time-lapse live-cell imaging. CRISPR-HiBiT BRD4 cells were treated with MZ1, a BET bromodomain degrader. Uniform loss of BRD4 was observed over 2 hours. Imaging was performed using an Olympus LV200 System.

Use of HiBiT for Protein Degradation

HiBiT is a popular tag for studying endogenous proteins. HiBiT is a small, 11 amino acid peptide that binds with high affinity to a larger subunit called LgBiT. The bound complex has high luciferase activity and will generate a bright luminescent signal in the presence of added substrate. The small size of the HiBiT tag allows for higher knock-in efficiency compared to larger tags, without the need for molecular cloning steps, making it well suited to CRISPR/Cas9 editing workflows.

Addition of compounds that elicit degradation results in loss of luminescence signal, which is highly quantitative and can be measured in real time. Cellular dose-response curves can be obtained and monitored over a 24- to 48-hour time frame, allowing for accurate determination of degradation rate, Dmax, DC50 values and protein recovery. This approach allows rapid rank ordering of degradation against a series of different parameters, and the assay is suitable for high-throughput screening.


krasg12c with lc-2 protac


kkrasg12c with MRTX849 protac

Live cell degradation kinetics of endogenous HiBiT-KRasG12C following PROTAC treatment. Panel A. HiBiT was inserted via CRISPR/Cas9 at the N-terminal endogenous KRasG12C loci in the MIA-PaCa2 cell line. Following LgBiT expression, dose-response kinetic degradation experiments were performed using the VHL-based KRasG12C PROTAC, LC-2, in CO2-independent medium containing Nano-Glo® Endurazine™ Substrate. Fractional RLU is plotted relative to the DMSO control. Panel B. Similar live-cell luminescent studies of HiBiT-KRasG12C in MIA-Paca2 cells using the parent inhibitor, MRTX849, which does not show loss of KRasG12C over the same dose-response treatments.

For a complete list of ready-to-use CRISPR knock-in reporter cell lines including WT and mutants for several KRAS, B-RAF and NRAS, see our current cell line list.

Protein:Protein Interactions with NanoBRET™ and NanoBiT® Technology

NanoBRET™ Technology

NanoBRET™ technology enables sensitive, reproducible detection of protein-protein interactions (PPI) in the natural cellular environment. The use of full-length proteins expressed at low levels enables PPI monitoring and screening studies that reflect true cellular physiology. The bright, blue-shifted donor signal and red-shifted acceptor create optimal spectral overlap, increased signal and lower background compared to conventional BRET assays.

NanoBRET Assay Principle


Principle of the NanoBRET™ PPI assay.

You can read more about the capabilities of NanoBRET™ technology and see example data in this publication: 
Machleidt, T. et al. (2015) NanoBRET—A novel BRET platform for the analysis of protein–protein interactionsACS Chem. Biol. 10(8), 1797–1804.


BRAF cRAF dimerization


KRas G12C/RAF NanoBRET assay data

NanoBRET™ assays across RAS and RAF family members for the study of inhibition and/or induction of protein interactions in the pathway. Kinetic RAS:RAF assays +/– BI-2852 inhibitor (Panel A); RAF dimerization assays showing induction with GDC0879 (Panel B).

NanoBiT® Technology

NanoLuc® Binary Technology (NanoBiT) is a two-subunit system based on NanoLuc® luciferase that can be applied to the intracellular detection of PPIs in live cells. This short video explains how the technology works.

nanobit ppi assay

Principle of the NanoBiT® PPI assay.

To learn more about using NanoBiT® technology to study PPIs involved in the RAS pathway, download this posterDetecting intracellular BRAF/CRAF dimerization using a novel luminescent complementation system.


NanoBiT® assays to monitor interaction of KRAS 4B wild-type and mutants with RAF isoforms
NanoBiT® assays to monitor interaction of KRAS 4B wild-type and mutants with RAF isoforms
NanoBiT® assays to monitor interaction of KRAS 4B wild-type and mutants with RAF isoforms

NanoBiT® assays to monitor interaction of KRAS 4B wild-type and mutants with RAF isoforms. KRAS 4B with the CRAF RBD domain (Panel A); KRAS 4B with full-length CRAF (Panel B); KRAS 4B with full-length BRAF (Panel C).

We have a variety of NanoLuc®, HaloTag® and BiBRET vectors to help you get started with your PPI assays. Contact us for more information.

Lumit™ Phospho-ERK Immunoassay

The Lumit™ Immunoassay Cellular System is a no-wash bioluminescent immunoassay that measures target analytes directly in cell lysates. No media removal or lysate transfer—just add labeled antibodies to the sample, add the detection reagent and read the luminescent signal—all in a single plate! We have customized this assay to detect phospho-ERK activity.


The principle of the Lumit™ Immunoassay Cellular System.


lumit immunoassay for erk activation and inhibition


lumit immunoassay for erk activation and inhibition

Activation and deactivation of the MAPK pathway. Panel A. Cultures containing 50,000 seeded MCF-7 cells were starved overnight. The cells were then untreated or treated with various concentrations of EGF for 5 minutes before phospho-ERK1 was measured by the Lumit™ Immunoassay Cellular System–Set 1 to determine the EGF EC50. Panel B. After starvation, 50,000 seeded MCF-7 cells were pretreated with various concentrations of MEK inhibitor, U0126, for 1 hour and then treated with EGF (30ng/ml, 5 minutes) before phospho-ERK1 was measured by the Lumit™ Immunoassay Cellular System–Set 1 to determine the potency of the inhibitor (IC50).

For more information, see our Application Note or visit the Lumit™ Immunoassay Cellular System product page.

GTPase-Glo™ Assay

The GTPase-Glo™ Assay analyzes the intrinsic activities of GTPase, GAP-stimulated GTPase, GAP and GEF. The assay measures enzymatic conversion of GTP remaining, after the GTPase reaction, into ATP followed by bioluminescent detection of the created ATP.

Principle of the GTPase-Glo assay

Principle of the GTPase-Glo™ Assay.

RAS GTPase assay data

GTPase activity and GAP-mediated GTPase activity of Ras and NF1-333. Reactions were assembled with 2μM wildtype or mutant Ras, 1μM NF1-333 and 5µM GTP in GTPase/GAP Buffer containing 1mM DTT. The final reaction volume was 10μl. Reactions were incubated for 90 minutes at room temperature. To the completed GTPase reactions, 10μl of GTPase-Glo™ Reagent was added and incubated for 30 minutes at room temperature. Detection Reagent (20µl) was added, plates were incubated for 5–10 minutes at room temperature and luminescence was recorded using the GloMax® Discover System.

For more information, see this publication, or visit the GTPase-Glo™ Assay product page.

Learn more about products and technologies for studying kinase pathways.