RhoGEF Exchange assay

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.

• Exchange Buffer
• His-tagged Cdc42 protein (Cat nr CD01)
• His-tagged Rac1 protein(Cat nr RC01)
• His-tagged RhoA protein (Cat nr RH01)
• His-tagged hDbs protein (Cat nr GE01)
• 384-well black half area round bottom plate
• 96-well black half area flat bottom plate
• Detailed instructions and troubleshooting manual.

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

Product Uses Include

• Determination of the activity and GTPase specificity of uncharacterized GEFs.
• Biochemical characterization of small GTPases and their associated GEFs.
• Examination of the regulation of GEF activity by different cofactors or protein domains.
• Screen the mutant protein of either GEFs or GTPases for activity and substrate specificity.
• Identification of GEF inhibitors in HTS (high throughput screen) format. Please inquire for significant discounts on large quantities of any reagents in this kit