View This Webinar on The Transcreener Method For Screening and Profiling GTPases

NRAS Assay - A Transcreener GDP Assay Application

Transcreener GDP Assay directly measures GDP produced by NRAS (Neuroblastoma Rat Sarcoma Virus Oncogene Homolog). These GDP measurements allow researchers to effectively determine the activity of the NRAS enzyme. The assay provides a powerful tool to screen compound libraries for NRAS modulators to help find new therapies for disease.

The kit comes complete with the detection reagents required to measure activity. NRAS enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

NRAS Enzyme Reaction Cycle

NRAS Assay On Off Schematic

How Does This NRAS Assay Work?

The Transcreener NRAS Assay determines NRAS enzyme activity by directly measuring the GDP formed by the enzyme using the Transcreener GDP Assay. By detecting GDP output, the assay provides a universal method to assess the activity of any GDP-producing enzyme in real time. 

Transcreener NRAS Assay technology uses a simple but highly effective method that consists of an antibody selective to GDP and a far-red fluorescent tracer. GDP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The NRAS assay is available with FP, FI, and TR-FRET. It is an easy-t0-use mix-and-read format. Perform your enzyme reaction, add the detection reagents, and measure. The simplicity of the system yields robust results that also make it extremely amiable to HTS.

Direct Detection of GDP to Measure NRAS Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener NRAS Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

Transcreener KRAS FI Readout

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

Transcreener KRAS Assay TR-FRET Readout

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of NRAS
  • Screen Compound Libraries for NRAS Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled GDP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The NRAS assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Detection of GDP Released By NRAS (EDTA Concentration Dependence)

EDTA enhances the GDP exchange and GTP hydrolysis of some GTPases. The assay conditions below are chosen to generate the best assay window and ability to study the enzyme. The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 1 µM GTP. Linearity is shown here under initial velocity conditions when raw data is converted to GDP formed. The enzyme buffer includes 20 mM Tris pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 5 μg/mL GDP antibody and 2 nM GDP Alexa 633 tracer. The NRAS enzyme reaction took place for 60, 120, and 180 minutes. The Detection Mix was added with the substrate to start the reaction and incubated at 30°C. Readings were taken with a CLARIOstar plate reader.

NRAS Enzyme Titration

NRAS Enzyme Titration Curve Concentration Dependence

Linear Response

NRAS Linear Response Curve Concentration Dependence

Optimal Assay Conditions: 125 nM NRAS for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion

Detection of GDP Released By NRAS (Time Dependence)

The assay demonstrates linearity when raw data is converted to GDP using a standard curve. Here we use 1 µM GTP. Linearity is shown here under initial velocity conditions when raw data is converted to GDP formed. The enzyme buffer includes 20 mM Tris pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 5 μg/mL GDP antibody and 2 nM GDP Alexa 633 tracer. 10 mM EDTA substrate was used based on the concentration dependent curve. The NRAS enzyme reaction took place for 60, 120, and 180 minutes. The Detection Mix was added with the substrate to start the reaction and incubated at 30°C. Readings were taken with a CLARIOstar plate reader.

NRAS Enzyme Titration

NRAS Enzyme Titration Curve Time Dependence

Linear Response

Optimal Conditions: 125 nM NRAS for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion

NRAS Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your NRAS program (right here in Madison, Wisconsin). We can provide modulator potency profiling for NRAS, along with a variety of other GTPase enzymes, related GAPs, GEFs, and other proteins. Get accurate IC50 results fast to understand how your lead molecule interacts with other GTPases. 

Assay Development Services

Contact Us to Learn More

Contact us today to see if BellBrook's NRAS profiling services will advance your research. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

The Role of NRAS As a Therapeutic Target

NRAS is part of the RAS GTPase family oncogenes, which have high mutation rates among several poor prognosis cancers. In the past, directly targeting RAS oncogenes in drug discovery seemed impossible; however, recent advancements in drugging RAS-target therapies show promise for therapeutic cancer treatments. With NRAS, there is hope for targeting several NRAS mutated malignancies, such as NRAS mutant metastatic melanoma (Boespflug et al, 2017).

The Transcreener GDP NRAS Assay is an excellent tool for researchers examining NRAS for therapeutic treatments.

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