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Screening for inhibitors of RGS protein GAP Activity using the Transcreener GDP Assay

GAP Assay - A Transcreener GDP Assay Application

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

The most direct way to detect RGS function is by measuring the increased GTPase activity of the associated Gα protein. However, the GTPase activity of isolated Gα proteins is limited by GDP dissociation, so RGS GAP activity is difficult to measure using simple biochemical assays. Use your own GAP protein or overcome this kinetic constraint of RGS function by using proprietary Gα protein variants with altered GTP hydrolysis and GDP dissociation rates that enable detection of RGS GAP activity using the Transcreener® GDP Assay.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

GAP Enzyme Reaction Cycle

GAP & GEF Enzyme Reaction Cycle

How Does This GAP Assay Work?

The Transcreener GDP Assay determines GAP activity by measuring increases in GDP formation as GAP 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 GAP Assay technology uses as 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  GAP 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 GAP Activity

Fluorescent Polarization (FP)

Transcreener GAP Assay Schematic

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

Fluorescent Intensity (FI)

Transcreener GAP 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 GAP Assay TR-FRET Readout

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

Applications

  • Measure GTPase-GAP Catalytic Activity
  • Screen Compound Libraries for GAP 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 GAP assay is compatible with 96, 384, and 1536-well formats. 

Mix-and-Read-GAP-Assay

GAP 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.

Assay Development 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

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Transcreener GDP Assay Kits - For GAP Activity Assays

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

Assay Plates for FP & FI

Assay Plates for TR-FRET

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. Members of the “regulator of G-protein signaling” (RGS)-protein superfamily act as GAPs for heterotrimeric G-protein alpha subunits, thereby attenuating GPCR signaling.

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

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