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

The Only Mix-and-Read Fluorescent GTPase Assay

The Transcreener GDP GTPase assay is a homogenous, fluorescent immunoassay that monitors the activity of GTPases along with GEF and GAP partners. The assay directly measures the amount of GDP produced in an in vitro GTPase reaction.

Learn More About Measuring GEF and GAP Activity:

Three Readouts Available

Three available fluorescent readouts provide a safe, HTS-compatible alternative to cumbersome radioassay methods and are more sensitive and less subject to interference than colorimetric phosphate detection methods such as malachite green. All readouts use far-red tracers to minimize compound interference and lower false-positive rates.

  • Fluorescence polarization (FP)
  • Fluorescence intensity (FI)
  • Time-resolved Forster-resonance-energy-transfer (TR-FRET)

GTPase Assay Principle - Universal GDP Detection

GTP hydrolysis from GTPase activity creates the product GDP and an additional phosphate. GDP displaces tracer from a highly selective monoclonal antibody resulting in a decrease in fluorescence polarization and TR-FRET or an increase in fluorescence intensity - perfect for your high throughput screen!

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

Assay Features

  • Easy-to-Use, Ultra-Sensitive GTPase Activity Assay
  • Homogenous, simple, mix-and-read method
  • Tunable dynamic range to match the target of interest
  • HTS Compatible with 96, 384, and 1536-well plates
  • Direct detection of GDP to monitor GTPase, GAP, and GEF Activity
  • Stable assay reagents – minimum 8-hour reagent and signal stability even at room temperature!

Single Step - Mix-and-Read

GDP GTPase Assay Schematic

Turnkey Protocol: Run enzyme reaction, add detection reagents, and read plates

Assay 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, including 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.

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

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

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 right here in Madison, Wisconsin will use their extensive biochemistry and enzymology expertise to work with you and accelerate 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.

Assay Development Services

Contact Us to Learn More

Contact us today to see if BellBrooks'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.

GTPases, GEFs, and GAPs as Drug Targets

GTPases act like switches and control a variety of cellular processes as they cycle between inactive (GDP bound) and active (GTP bound) conformations.  GTPases form a relatively large family of enzymes, all of which are characterized by the ability to hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP).

Many cellular processes are modulated by GTPases including actin cytoskeletal organization, cell growth, proliferation, differentiation, and survival. For these reasons, GTPases have become a target for many diseases including cancer, Alzheimer’s and Parkinson’s.

Small GTPases include the Ras, Rho, Rab, Arf, and Ran subgroups. Mutant K-Ras proteins are significant drivers of human cancers and may play a direct role in about one million cancer cases per year. Ras GTPases are important targets in drug discovery and development, as genetic alterations in Ras GTPase are very common in human cancers.

GTPase Activity Assay On Off Cycle

GTPase Cycle - ‘molecular switch’. Bound GTPase-GDP and inactive form with GTPase-GTP as the active.

Rho family GTPases control cell growth, movement, and gene expression and are often misregulated in cancer pathways, especially cell migration and invasion. However, Rho GTPases are infrequently mutated and their tumorigenic functions are often mediated by overexpression of GEFs which positively regulate.

Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow the formation of the active, GTP-bound complex. Development of inhibitors that specifically disrupt GEF action on a target Rho GTPase may be a promising therapeutic strategy for blocking Rho-dependent oncogenesis.

Unlike GTP binding assays, the Transcreener method is based on multiple catalytic cycles for both the GEF and the GTPase with an unmodified GTP substrate making it is a more direct measure of GEF’s intrinsic functional activity.

Members of the regulator of G-protein signaling (RGS) superfamily have emerged as critical modulators of specific GPCR signal transduction pathways. Via their conserved RGS domain that confers GTPase-activating protein (GAP) activity, RGS proteins attenuate GPCR signaling by deactivating heterotrimeric G-protein alpha subunits. The steady-state GTPase activity of isolated Gα proteins is limited by GDP dissociation, so steady-state hydrolysis cannot be used to measure GAP activity in simple biochemical assays. To overcome this hurdle, mutated Gα proteins with decreased GTP hydrolysis rates and increased GDP dissociation rates are required. Mutated Gα proteins and RGS domains can then be coupled with the homogenous, fluorescence-based Transcreener GDP assay to demonstrate an HTS-compatible assay system for selective modulation of GPCRs.

A Guide to Navigating Hit Prioritization After Screening Using Biochemical Assays

So you have performed your screen. What’s next? This guide is focused on how biochemical assays are used for characterizing and prioritizing compounds following a primary screen with an enzyme target, whether using high throughput screening (HTS) or virtual screening (VS). A typical screening funnel is shown below, with the many applications of the biochemical activity assay highlighted.

Here we discuss strategies for hit-to-lead selection including:

  • Assay Considerations
  • Hit Confirmation
  • Running a Dose-Response
  • Compound Triaging
  • Hit Expansion
  • Mechanism of Action Studies
  • Residence Time Measurements
A Guide to Navigating Hit Prioritization After Screening Using Biochemical Assays

Enter your contact info and receive hit triaging tips with this 9-page guide!