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

RhoA Assay - A Transcreener GDP Application

Transcreener GDP Assay directly measures GDP produced by RhoA (Ras homolog family member A). These GDP measurements allow researchers to effectively determine the activity of the RhoA enzyme. The assay provides a powerful tool to screen compound libraries for RhoA modulators to help find new therapies for disease.

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

RhoA Enzyme Reaction Cycle

RhoA Assay On Off Schematic

How Does This RhoA Assay Work?

The Transcreener RhoA Assay determines RhoA 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 RhoA 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 RhoA assay is available with FP, FI, and TR-FRET readouts. It is in an easy-to-use mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of GDP to Measure RhoA Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener RhoA 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 RhoA
  • Screen Compound Libraries for RhoA 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 RhoA assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Detection of GDP Released By RhoA (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 RhoA 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.

RhoA Enzyme Titration

RhoA Enzyme Titration Concentration Dependence

Linear Response

Linear Response Curve RhoA Assay Concentration Dependence

Optimal Assay Conditions: 65 nM KRAS for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion

Detection of GDP Released By RhoA (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 RhoA 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.

RhoA Enzyme Titration

RhoA Enzyme Titration Time Dependence

Linear Response

Linear Response Curve RhoA Assay Time Dependence

Optimal Assay Conditions: 65 nM RhoA for 2 hours with 10 mM EDTA achieves >150 mP assay window and 20% conversion

RhoA 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 RhoA program (right here in Madison, Wisconsin). We can provide modulator potency profiling for RhoA, 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 RhoA 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 RhoA As a Therapeutic Target

RhoA is a GTPase enzyme that regulates cell morphology phenotypes, cell polarity, and cell migration. Recently, RhoA was proven to be a target for small molecule inhibitors, gaining interest for cancer therapeutics. RhoA is now a target for several RhoA related cancers, such as gastric cancer (Nam et al, 2019).

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

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