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RhoGAP 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”, which mediates signals downstream. 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).

Small G-proteins of the Ras superfamily typically have an inrinsic GTPase activity, meaning that after they get activated (GTP-bound) by GEFs, they eventually “turn themselves off”. The intrinsic GTPase rate, however, is normally very slow, so the “automatic” turning off process can take up to hours.

Because of the slow intrinsic GTPase activity in most Ras superfamily GTPases, their GAPs are important signaling molecules that function to terminate their signaling. GAPs function by binding to the active, GTP-bound, form of their target G-proteins and greatly stimulating the G-protein’s intrinsic GTPase activity, thereby quickly turning off the G-protein.

BK105

This GAP assay kit measures the amount of inorganic phosphate (Pi) that is produced as a result of G-protein dependent hydrolysis of GTP to GDP + Pi. The kit contains all the reagents needed for the assay including GTPases, a positive control RhoGAP, carefully optimized buffers and phosphate detection reagent. The assay can be performed in either 96-well plates or in 384-well plates. There is enough reagents for 80 assays (20 for each provided GTPase) in the 96-well format and 160 assays (40 for each provided GTPase) in the 384-well format

Kit contents

The kit contains sufficient materials for 80-160 assays.

The following components are included:
Equipment needed

96-well or 384-well plate spectrophotometer capable of measuring absorbance at 650 nm wavelength

Example results

Figure 1 in the gallery shows the GTPase activity of RhoA with and without the addition of the catalytic domain of p50RhoGAP.

Product Uses Include


For a kit to measure GEF activity, see Cat.nr BK100.

 

 


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