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RhoGEF Exchange assay

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Introduction

The Ras superfamily of small GTPases (such as Ras, Rho, Rab, Arf and Ran proteins) serve as binary switches cycling between a GDP-bound “OFF state” and a GTP-bound “ON state”. In cells, the cycling between the two states is mainly controlled by two types of regulatory proteins: the activating Guanine nucleotide Exchange Factors (GEFs) and the inactivating GTPase Activating Proteins (GAPs).

GEFs function by catalyzing the exchange of nucleotide on their target GTPases. The GEF will bind to a nucleotide-bound GTPase, which causes the bound nucleotide to be released, thus resulting in a nucleotide free GEF-GTPase reaction intermediate. This nucleotide free complex will then take up a new nucleotide after which the GEF is released from the GTPase. Because GEFs typically have a higher affinity for GDP-bound GTPases than for the corresponding GTP-bound GTPase and the intracellular ratio of GTP to GDP is about 10:1, GEFs will drive the exchange from GDP-bound GTPases to GTP-bound GTPases.

BK100

The fluorophore based assay in this kit is suitable for measuring nucleotide exchange on GTPases in either 96-well or 384-well format. The kit measures the uptake of the fluorescent nucleotide analog N-methylanthraniloyl-GTP (mant-GTP) into GTPases. The uptake can be measured due to the spectroscopic difference between free and GTPase-bound mant-GTP. As mant-GTP gets bound in the nucleotide binding pocket of a GTPase, its fluorescence (ex: 360 nm, em: 440 nm) increases dramatically (1, 2). Therefore, the enhancement of mant-GTP fluorescent intensity in the presence of a small GTPase indicates nucleotide uptake (or exchange for already bound nucleotide) by the GTPase.

Kit contents

The kit contains enough materials for 60 assays in 96-well format or 300 assays in 384-well format.

The following components are included:
Dbs is an effective GEF for Cdc42 and RhoA but shows only very low GEF activity for Rac1 (see Figures 1 & 2 in the gallery).

Equipment needed

Fuorescence plate reader (96 or 384-well) capable of measuring fluorescence at ex: 360 nm, em: 440 nm

Product Uses Include

 

 For a kit to measure GAP activity, see Cat nr BK105

 


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