Targeting LRRK2 for Parkinson’s Disease with the Transcreener® GDP GTPase Assay

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Transcreener GDP GTPase Assay Kit

Transcreener GTPase Assay Kit directly measures GDP produced by GTPase reactions as they hydrolyze GTP. Use this assay for quick and simple high-throughput screening of GTPases, along with GAPs and GEFs. The generic nature of the assay simplifies compound and inhibitor profiling for multiple GTPase targets.

The kit comes complete with the detection reagents required to measure activity. The inhibitor screening kit does not include the GTPase enzyme. Please contact us for questions related to acquiring the enzyme.

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

Learn More About Measuring GEF and GAP Activity:

How Does The GTPase Assay Work?

The Transcreener GDP Assay measures GTPase enzyme activity by directly measuring the GDP produced during GTP hydrolysis. By detecting GDP output, the assay provides a universal method to assess the activity of any GDP-producing enzyme in real-time.

Transcreener GTPase 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 GTPase 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.

GTPase Enzyme Reaction Cycle

Target Applications

Click on the respective target to learn more.

GTPase Family	Enzyme
Ras Family	KRAS
	HRAS
	NRAS
	RRAS
Rho Family	RhoA
	RhoC
	Cdc42
	Rac1
Ran Family	Ran

Direct Detection of GDP to Measure GTPase Enzymatic Activity

Fluorescent Polarization (FP)

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

Learn More About The FP Assay

Transcreener GDP FP Assay Tech Manual

Fluorescent Intensity (FI)

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

Learn More About The FI Assay

Transcreener GDP FI Assay Tech Manual

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

Transcreener GTPase Assay TR-FRET Readout

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

Learn More About The TR-FRET Assay

Transcreener GDP TR-FRET Assay Tech Manual

Applications

Measure enzymatic activity of GTPase
Screen compound libraries for GTPase Modulators
Quantify Inhibitor Potency
Inhibitor Selectivity Profiling
Measure Drug-Target Residence Time

What Can You Do With Transcreener

Measure guanine nucleotide exchange factor (GEF) activity
Measure GTPase-activating proteins (GAP) activity
Detect any GDP producing enzyme: GTPases, Gα proteins, Ras-like G proteins (Ras, Roc, Rac, cdc42, etc.), fucosyltransferases, mannosyltransferases

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

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

Features

Direct detection of GDP to monitor GTPase, GAP, and GEF activity
Easy to use, homogenous, one-step format
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method (no disposal concerns)
Stable assay reagents- minimum 8-hour reagent and signal stability even at room temperature!
More sensitive and less subject to interference than colorimetric phosphate detection methods such as malachite green

Tunable to Your Target

High Quality Data Under Initial Velocity Conditions

GTPase Assay Data Under Initial Velocity Conditions

Standard curves mimicking the enzymatic conversion of GTP to GDP using the Transcreener GDP FP Assay. The dynamic range of the assay can be easily tuned for different initial GTP concentrations. Excellent Z' values are achieved even at low GDP reagent formation.

Use with a Variety of GTPase Enzymes

Based on BellBrook Lab's proprietary nucleotide immunodetection technology, the Transcreener GDP Assays are compatible with any enzyme class that produces GDP. This includes monomeric small G proteins, such as CDC42, and Gα subunits of heterotrimeric G proteins, such as Gαi1. Learn how Transcreener can be used to study targets like Cdc42 and RhoA in Springer's Rho GTPases Methods and Protocols. Discover how researchers at UT Southwestern used the GDP assay to screen dynamin GTPase activity.

GTPase Enzyme Titration with Cdc42 and RhoA

Cdc42 and RhoA GTPase titration using the Transcreener GDP Assay with an FP readout

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GTPase Profiling Services

Do you want to advance your program without performing an assay in-house? Our scientists can assist you!

At BellBrook, we have experts in biochemistry and enzymology that will collaborate with you to expedite your GTPase program. We can provide inhibitor potency profiling for a variety of GTPase enzymes and related proteins. Get accurate IC₅₀ results fast, to understand how your inhibitor interacts with other GTPases.

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.

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

Instrument	FP Assays	FI Assays	TR-FRET Assays
TriStar²S LB 942	In Review	Validated	Validated
Tristar 5	In Review	Validated	In Review
Mithras² LB 943	In Review	Validated	Validated

Cytation^TM 5	Validated	Validated	Validated
Cytation^TM 3	Validated	Validated	Validated
Cytation^TM 1	Validated	Validated	Validated
Synergy^TM H1	Validated	Validated	Validated
Synergy^TM 2/H4/4	Validated	Validated	Validated
Synergy^TM HTX	Not Capable	Validated	Not Capable
Synergy^TM Neo 2	Validated	Validated	Validated

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^TM	Validated	Validated	Validated

Sense	In Review	Validated	In Review

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	Validated	Validated	Validated
SpectraMax^® i3x	Validated	In Review	Validated

EnVision^®/EnVision^® Xcite	Validated	Validated	Validated

Infinite^® M1000/M1000Pro/Safire2^TM	Validated	Validated	Validated
Infinite^® M200	Not Capable	Validated	Not Capable
Infinite^® F500	Validated	Validated	Validated
Infinite^® F200/Ultra^evolution	Contact Us	Validated	Contact Us
Spark^TM 10M	Validated	Validated	Validated

What's Included


Component	Notes
GDP Antibody	The concentration of GDP Antibody needed for an enzyme target is dependent upon the GTP concentration and buffer conditions in the enzyme reaction. Sufficient antibody is included in the kit to complete 1,000 assays or 10,000 assays at a GTP concentration up to 100 µM.
GDP Tracer	When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format.
Stop & Detect Buffer B	The Stop & Detect Buffer B components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction.
GTP	The GTP supplied in this kit can be used for the enzyme reaction and to create a GDP/GTP standard curve, if desired.
GDP	GDP is used to create the GDP/GTP standard curve.

What You Will Need


Component	Notes
GTPase Enzyme	Please use purified enzymes for the best results.
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl₂ or MnCl₂, Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates (Cat. #4514). FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates (Cat. #4514) and Corning 96-well, half-area plates (Cat. # 3686). TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates (Cat. #4513).
Plate Reader	A multi-detection microplate reader configured to measure output of the GDP tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 0.5 µL into 384-well plates.
Ultrapure Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substances and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

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