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

Transcreener GDP Assay (TR-FRET) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener GDP Assay - TR-FRET Readout

The Transcreener^® GDP Assay with TR-FRET readout uses displacement of a tracer by GDP to measure enzymatic activity. This disrupts FRET from the antibody-bound Terbium chelate leading to a decrease in the TR-FRET signal. In addition to the time-resolved signal, the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.

The assay produces an excellent signal at low substrate conversion for a broad range of initial GTP concentrations (0.1-1,000 µM). Both reagents & signal are stable at room temperature for over 24 hours, providing outstanding flexibility for automated high-volume screening programs. The universal detection method allows screening of virtually any GTPase in a high throughput format.

Used in over millions of well by pharma since 2006, Transcreener is the most extensively validated GDP detection method available backed by the best technical support in the industry.

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.

Direct Detection of GDP to Measure GTPase Enzymatic Activity

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

Learn More About the Transcreener GDP Assay

Transcreener GDP TR-FRET Assay Tech Manual

Applications

Measure enzymatic activity of GTPases
Screen compound libraries for GTPase modulators
Quantify inhibitor potency
Inhibitor selectivity profiling
Measure drug-target residence time

Features

Direct detection of unlabeled GDP
Robust Assay Z' > 0.7 under initial velocity conditions
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method
Use with GTP concentrations from 0.1 to 1000 µM
Universal - use with virtually any GTPase

Easy-to-Use, Mix-&-Read, HTS Ready

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

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Example Applications

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

Far Red TR-FRET Readouts Validated on Major Multimode Readers

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

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

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Validated
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Validated
	SpectraMax^® Paradigm	Contact Us
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	In Review

	EnVision^®/EnVision^® Xcite	Validated

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

Validation Criteria for Plate Readers

384-Well Format
Z'-Factor ≥ 0.7 at 10% conversion of 10 μM ATP
Z' specifications to be met using Transcreener ADP² Assay Reagents
Read time to achieve Z' specifications ≤ 5 minutes

Look for these stickers on instruments that have successfully met the validation criteria.

The instrument validation program is a collaborative effort with the major suppliers of multimode readers to insure that Transcreener assays perform optimally on your specific instrument. Scientists at BellBrook and/or the instrument vendor have tested Transcreener reagents with each instrument to determine optimal filters and settings for maximal assay performance. We provide you with application notes with all the important details, so you can be confident of success using Transcreener assays on the instrument of your choice.

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What's Included


Component	Notes
GDP HiLyte647 Tracer	The concentration of GDP HiLyte647 Tracer needed for an enzyme target depends upon the GTP concentration and buffer conditions in the enzyme reaction. Sufficient tracer is included in the kit to complete 1,000 assays (Part #3021-1K) or 10,000 assays (Part #3021-10K) at a GTP concentration up to 100 µM GTP.
GDP Antibody-Terbium Conjugate	The final antibody concentration in the reaction is 4 nM in a 20 µL final reaction volume.
Stop & Detect Buffer C, 10X	The Stop & Detect Buffer C 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. The final concentration of Stop & Detect Buffer C at the time of FRET measurement is 0.5X.
GTP	The GTP supplied in the 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	Transcreener GDP assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphates and nucleotidases, can produce background signal and reduce the assay window.
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl₂ or MnCl₂, EGTA, Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. It is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning^® 384-well plates (Cat. #4513).
Plate Reader	A microplate reader configured to measure TR-FRET of the Tb:HiLyte647 donor:acceptor pair is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL into 384-well plates.
Ultrapure Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

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Transcreener GDP Assay Kits - TR-FRET Readout

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

Cat. No.	Product Description	Price	Add to Cart
3021-1K	Transcreener GDP TR-FRET Assay (1,000 Assay, 384 Well)	$639.00	Add to Cart
3021-10K	Transcreener GDP TR-FRET Assay (10,000 Assay, 384 Well)	$2,925.00	Add to Cart
3021-100K	Transcreener GDP TR-FRET Assay (10 X 10,000 Assay, 384 Well)	$21,938.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price	Add to Cart
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$55.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$65.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$529.00	Add to Cart

Transcreener® GDP Assay Kit – TR-FRET Readout

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

Transcreener GDP Assay (TR-FRET) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener GDP Assay - TR-FRET Readout

Direct Detection of GDP to Measure GTPase Enzymatic Activity

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

Applications

Features

Easy-to-Use, Mix-&-Read, HTS Ready

Back To Top

Example Applications

Far Red TR-FRET Readouts Validated on Major Multimode Readers

Validation Criteria for Plate Readers

Back To Top

What's Included

What You Will Need

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Transcreener GDP Assay Kits - TR-FRET Readout

Assay Plates for TR-FRET

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Related Resources

Researchers Employ GTPase Assay To Investigate Rab Proteins

High Throughput Screenings For Drug Discovery

Detecting Rho-GEF Activity Using the Transcreener® GDP Assay

Researchers from Bellbrook Labs Pen Chapter in Springer's Rho GTPases: Methods and Protocols

Posters and Presentations

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