The Transcreener GTPase Assay Kit relies on direct detection of GDP produced by GTPases during GTP hydrolysis. The direct detection approach is a novel method to quantify activity of GTPases and their stimulation by related GAP and GEF proteins. Direct detection simplifies the protocol (no coupling enzyme needed) and minimizes compound interference.
Image Credit: BellBrook Labs
Because the method is based on direct GDP detection, it is compatible with most GTPase targets. It is an easy-to-use and HTS-ready assay designed for screening and profiling inhibitors. The assay can be used to:
- Measure the enzymatic activity of GTPases
- Screen compound libraries for GTPase modulators
- Quantify inhibitor potency
- Inhibitor selectivity profiling
- Measure drug-target residence time
GTPase Target Applications
The table below includes example GTPases we have validated with the Transcreener GDP Assay. Click on a target to learn more.
Source: BellBrook Labs
Why Choose Transcreener?
- Direct detection of GDP for monitoring GTPase, GAP, and GEF activity
- Easy to use, two-step format (no coupling enzymes needed)
- Far-red fluorescent readouts reduce compound interference
- A non-radioactive, safe technique
- Assay reagents are stable for at least eight hours, even at room temperature
- Greater sensitivity and less susceptibility to interference in comparison to colorimetric phosphate detection techniques like malachite green
- Available in an FP, FI, or TR-FRET readout depending on preference and plate reader compatibility
Example Data Using KRAS GTPase
Detecting GDP Under Initial Velocity Conditions with Varying EDTA Concentrations
KRAS enzyme titration with varying EDTA concentrations using the Transcreener GDP FP Assay. Assay conditions include 2 µM GTP, 20 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 1X Stop & Detect Buffer B, 10 μg/mL GDP antibody, and 4 nM GDP Alexa 633 tracer.
Image Credit: BellBrook Labs
KRAS linear response with varying EDTA concentrations using the Transcreener GDP FP Assay. The assay demonstrates linearity when raw data is converted to GDP using a standard curve.
Image Credit: BellBrook Labs
Detecting GDP Under Initial Velocity Conditions with Varying Enzyme Reaction Times
KRAS enzyme titration with varying enzyme reactions time (60 min, 120 min, and 180 min) using the Transcreener GDP FP Assay. Assay conditions include 2 µM GTP, 20 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. The Detection mix included 1X Stop & Detect Buffer B, 10 μg/mL GDP antibody, and 4 nM GDP Alexa 633 tracer.
Image Credit: BellBrook Labs
KRAS linear response with varying enzyme reactions time (60 min, 120 min, and 180 min) using the Transcreener GDP FP Assay. The assay demonstrates linearity when raw data is converted to GDP using a standard curve.
Image Credit: BellBrook Labs
GTPase Profiling Services
BellBrook Labs also offers GTPase profiling services for scientists who want to move their program forward, but don't want to bring an assay in-house. This can include testing compounds at a single concentration or in dose-response mode for the determination of IC50 values. Their services feature customizable assay conditions, quick turnaround times, and direct collaboration with a BellBrook scientist.