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

Transcreener GDP Assay (FP) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener GDP Assay Kit - FP Readout

The Transcreener GDP Assay with FP readout measures GDP via antibodies selective to GDP and a far-red fluorescent tracer. GDP produced in the reaction competes with the tracer, decreasing fluorescent properties and providing a fluorescent readout. The universal detection method allows screening of virtually any kinase in a high throughput format.

The Transcreener Assay offers reagent stability and compatibility with commonly used multimode plate readers. The FP GDP Assay accommodates GTP concentrations ranging from 1 µM to 1,000 µM with excellent data quality (Z' ≥ 0.7) at low substrate conversion (typically 10-30%).

Used in over millions of wells 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

Fluorescence Polarization (FP)

Learn More About the Transcreener GDP Assay

Transcreener GDP FP 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 1 µM to 1,000 µM
Stable Assay reagents - minimum 8-hour reagent and signal stability even at room temperature!

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.

Example Applications

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

Detection of GDP Released by KRAS (FP Readout)

Below is an example of the Transcreener GDP Assay with FP readout detecting GDP released by the GTPase, KRAS. The assay demonstrates linearity when raw data is converted to GDP using a standard curve.

KRAS Enzyme Titration

KRAS Assay Enzyme Titration Curve Concentration Dependence

Linear Response

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Far Red FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	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	Not Capable
	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	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	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
Δ mP ≥ 95 mP at 10% conversion of 10 μM ATP
Z' and Δ mP specifications to be met using Transcreener ADP² Assay Reagents
Read time to achieve Z' and Δ mP 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 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 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 (Cat. #3009-1K) or 10,000 assays (Cat. #3009-10K).
GDP Alexa Fluor^® 633 Tracer	When displaced, changes in fluorescence can be read in an FP readout. The final tracer concentration in the 20 µL reaction is 2 nM.
Stop & Detect Buffer B, 10X	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. The final concentration of Stop & Detect Buffer B at the time of FP 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 phosphatases or 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, 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.
Plate Reader	A multidetection microplate reader configured to measure FP of the Alexa Fluor^® 633 tracer 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 - FP 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
3009-1K	Transcreener GDP FP Assay (1,000 Assay, 384 Well)	$559.00	Add to Cart
3009-10K	Transcreener GDP FP Assay (10,000 Assay, 384 Well)	$2,749.00	Add to Cart
3009-100K	Transcreener GDP FP Assay (10 X 10,000 Assay, 384 Well)	$20,618.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	Add to Cart