A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay

GEF Assay - A Transcreener GDP Assay Application

Transcreener GDP assay directly measures GDP produced by GTPases when GEF proteins stimulate the enzyme. These GDP measurements allow researchers to effectively determine the GEF activity. The assay provides a powerful tool to screen compound libraries for GEF modulators to help find new therapies for disease.

Active Rho GTPases stimulate cell growth and migration, sometimes triggering tumorigenesis or invasion. Rho GEFs (guanine nucleotide exchange factors) positively regulate Rho GTPases by accelerating GDP dissociation and allowing the formation of the active GTPase-GTP complex.

By accelerating GDP dissociation, the rate-limiting step of the GTPase catalytic cycle, GEFs enhance the steady state rates of GTP hydrolysis by GTPases. These changes can be detected using the Transcreener® GDP GEF Assay, which relies on a highly selective antibody and far-red fluorescent tracer to allow homogeneous detection of GDP with fluorescence readouts.

GEF Enzyme Reaction Cycle

GAP & GEF Enzyme Reaction Cycle

How Does This GEF Assay Work?

The Transcreener GEF Assay determines GEF activity by measuring increases in GDP formation as GEF proteins stimulate the GTPase. By detecting GDP output, the assay provides a universal method to assess the activity of any GDP-producing enzyme in real-time. 

Transcreener GEF 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 GEF assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also make it extremely amiable to HTS. 

Direct Detection of GDP to Measure GEF Activity

Fluorescent Polarization (FP)

Transcreener GEF Assay Schematic

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

Fluorescent Intensity (FI)

Transcreener GEF 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 GEF Assay TR-FRET Readout

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

Applications

  • Measure GTPase-GEF Catalytic Activity
  • Screen Compound Libraries for GEF Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

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 GEF assay is compatible with 96, 384, and 1536-well formats.

Mix-and-Read-GEF-Assay

Development of GEF Assays with Transcreener's GDP Assay

GTPase Titration

Cdc42 and RhoA titration to identify a concentration that produces ~20% of the maximal signal. The data indicates 39 nM Cdc42 & 78 nM RhoA are optimal for detecting GEF-dependent stimulation of steady-state GTPase activity.

Enzyme Titration GEF Assay

Dbs (GEF) Titration

Adding Dbs to reaction containing a low concentration of RhoA and Cdc42 created a dose-dependent increase in the signal observed, suggesting that accelerating GDP dissociation increases the steady-state rate of GTPase activity. Optimal Dbs concentration was 100 nM.

Dbs Titration2

GEF Effect on Rho GTPases

Time course data showing the change in GTPase activity of Cdc42 and RhoA in the presence and absence of Dbs, as measured by changes in FP using Transcreener® GDP assay. The polarization values from the GTPase/GEF reactions were converted to GDP formed.

Change in Cdc42 Activity With and Without Dbs

Continous GEF Assay With Cdc42 and Dbs
Standard Curve GEF Assay With Cdc42 and Dbs

Assay demonstrates linearity when raw data is converted to GDP using a standard curve

Change in RhoA Activity With and Without Dbs

Continous GEF Assay With RhoA and Dbs
Standard Curve GEF Assay With RhoA and Dbs

Assay demonstrates linearity when raw data is converted to GDP using a standard curve

GEF 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 GTPase program (right here in Madison, Wisconsin). We can provide modulator potency profiling for GTPases, 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.

Scientists Performing Lead Drug Discovery Services

Contact Us to Learn More

Contact us today to see if BellBrook's GTPase 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
bioteklogo 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
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

Transcreener GDP Assay Kits - For GEF Activity Assays

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

GAPs & GEFs As Therapeutic Targets

GTPases control diverse cellular processes by cycling between inactive GDP- and active GTP-bound conformations. GAPs & GEFs regulate this active and inactive cycling. Rho family GTPases control cell growth, movement and gene expression and are often misregulated in cancer pathways, especially cell migration and invasion.

Rho GTPases are infrequently mutated, and their tumorigenic functions are often mediated by overexpression of GEFs.  This positively regulates Rho GTPases by accelerating GDP dissociation to form the active, GTP-bound complex.

The Transcreener GDP GEF Assay is an excellent tool for researchers examining GEF for therapeutic treatments by directly measuring GDP produced in the reaction.

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