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

Transcreener GDP GTPase Assay

Transcreener GDP GTPase Assay directly measures GDP produced by in vitro GTPase reactions. This allows researchers to effectively determine the activity of the GTPase enzyme, along with GEF and GAP partners. The assay provides a powerful tool to screen compound libraries for GTPase modulators to help find new therapies for disease. 

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

Learn More About Measuring GEF and GAP Activity:

How Does The GTPase Assay Work?

The Transcreener GDP Assay determines GTPase enzyme activity by directly measuring the GDP formed by the enzyme 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

GTPase Assay On Off Schematic

GTPase Profiling Assay - Applications

GTPase FamilyEnzyme
Ras FamilyKRAS
HRAS
NRAS
RRAS
Rho FamilyRhoA
RhoC
Cdc42
Rac1
Ran FamilyRan

Direct Detection of GDP to Measure GTPase Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener GTPase Assay Schematic

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

Fluorescent Intensity (FI)

Transcreener GTPase 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 GTPase Assay TR-FRET Readout

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

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

GDP GTPase Assay Schematic

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

Assay Tunable to Your Target

GTPase Standard Curve in GTP Hydrolysis

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

GTPase Profiling 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 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.

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

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo 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
VANTAstar 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

The Role of GTPases As Therapeutic Targets

GTPases are highly conserved regulatory proteins involved in diverse roles such as signal transduction, protein biosynthesis, cell adhesion, membrane remodeling and trafficking, cytoskeletal reorganization, apoptosis, autophagy, and cell motility. GTPases have such extensive function in cellular operation that they are called master regulators of intracellular membrane traffic (Stenmark 2009).

GTPase inhibitors are attractive drug discovery targets. Because of their biological roles, GTPase inhibitors are explored for use in a multitude of diseases, such as cancers and neurodegenerative diseases. Finding therapeutic methods to reign in overactivity requires selective technologies that can help assess the ability to interact with the target.

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

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