RhoGAP assay (BK105)

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80-160 assays (Cat. # BK105) +$550.00

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Product Uses Include

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

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.

This GAP assay kit measures the amount of inorganic phosphate (P_i) that is produced as a result of G-protein dependent hydrolysis of GTP to GDP + P_i. 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

While the kit comes with Cdc42, Rac1, RhoA and Ras proteins, it can also be used for any other Ras superfamily GTPase. See our G-protein product family web page for other GTPases available from Cytoskeleton.

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

Kit contents
THe kit contains sufficient materials for 80-160 assays. The following components are included:

Reaction Buffer
His-tagged Cdc42 protein (Cat. # CD01)
His-tagged Rac1 protein(Cat. # RC01)
His-tagged RhoA protein (Cat. # RH01)
His-tagged Ras protein (Cat. # RS01)
GST-tagged p50RhoGAP, catalytic domain (Cat. # GAS01)
GTP stock (Cat. # BST06).
CytoPhos™ reagent.
96-well clear half area flat bottom plate
Detailed instructions and troubleshooting manual.

Equipment needed

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

Example results
The GTPase activity of RhoA with and without the addition of the catalytic domain of p50RhoGAP was tested (Fig 1).


	Figure 1. RhoGAP activity measured as GTP hydrolysis by RhoA protein. Each reaction contained reaction buffer + GTP with the addition of RhoA alone (RhoA), RhoGAP alone (GAP), or RhoA + RhoGAP (RhoA + GAP). Reactions were incubated at 37°C for 20 min. Phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos™ reagent and reading of absorbance at 650 nm.

RhoGAP assay (BK105)

RhoGAP assay (BK105)

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