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High Throughput GEF (Guanine nucleotide Exchange Factor) Assay Using BBL's Transcreener® GDP Assay
An Application Note is published to serve as a guide for using BellBrook Labs' Transcreener® GDP Assay to detect the activity of guanine nucleotide exchange factors (GEFs) based on their stimulation of steady state GTPase activity.
By: MoBiTec GmbH
The BellBrook Labs Transcreener GDP HTS Assays rely on direct immunodetection of GDP with a far-red readout in a simple mix-and-read format. The assays are available in three different detection modes: fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Forster resonance energy transfer (TR-FRET).
Monomeric small Ras/Rho GTPases serve as molecular switches by cycling between an inactive GDP-bound conformation and an active GTP-bound conformation. Activated GTPases generally stimulate cell growth and migration, and constitutively activated mutants can be oncogenic (e.g., K-Ras). Small GTPases generally bind GDP and GTP with extremely high affinity (dissociation constants in the nanomolar range or lower), and GDP dissociation is usually the rate-limiting step in the GTPase catalytic cycle. The role of GEFs, therefore, is to bind to GTPases and accelerate the dissociation of GDP, enabling regeneration of the GTP-bound activated form of the protein.
Here is shown by BBL an example of how the Transcreener GDP Assay can be used to measure the GEF-dependent stimulation of steady-state GTP hydrolysis activity of small GTPases using Dbs as a GEF for CDC42 and RhoA.
The general process for GEF assay development is comprised of the following three steps:
1. Select a concentration of GTPase that produces a small but clearly detectable signal in the Transcreener GDP Assay.
2. Titrate in the GEF to determine the concentration that yields a robust signal in the linear velocity region (<20% conversion of GTP to GDP).
3. Confirm the assay window at the optimal GTPase and GEF concentrations and perform assays alongside control reactions lacking GEF to indicate the presence of non-specific inhibitors.
The complete Application Note is available at:
Customers from Europe are welcome to visit www.mobitec.com or contact MoBiTec GmbH for Transcreener®
Custom quotes are available for bulk orders.
About Bellbrook Labs
BellBrook Labs is dedicated to providing the pharmaceutical industry with enabling screening tools to accelerate the discovery of more effective therapies. As a small, R&D-driven company, BBL identifies areas where there is a strong need but a significant technical hurdle, and then BBL develops a solution. The company is focused on taking creative
approaches to new product development and to continually improving the level of service provided to our customers. BellBrook is undergoing a big change with the launch of the first two iuvo™ products and the Invasion Assay Service in 2010. The company has built its reputation on Transcreener®
About MoBiTec GmbH
MoBiTec GmbH (Goettingen, Germany) is a privately held company (founded in 1987) that offers research tools for molecular and cell biology. Products include DNA vectors for cloning and expression, cell transfection reagents and cell culture tools, immobilized and soluble enzymes, products for genomics and proteomics research, numerous antibodies and recombinant proteins, superior fluorescence reagents and kits, affinity chromatography products, as well as general laboratory equipment.
In parallel to its own product lines, MoBiTec distributes products from international companies in Germany. MoBiTec products are distributed worldwide, in Germany from their home office, in other countries by distributors.