GTPase-Glo™ Assay

Simple, Sensitive GTPase Activity Assay

Simple add-and-read format
Suitable for 96-, 384- and 1536-well plates
Low background and large dynamic range

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Size

1,000 assays 10,000 assays

Catalog Number: V7681

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Catalog Number: V7682

Please Enquire

Bioluminescent Detection of GTPase, GAP and GEF Activities

The GTPase-Glo™ Assay is a homogeneous, bioluminescent assay that is used to measure the activities of GTPases and their associated proteins (GEFs and GAPs) in vitro. The assay quantitates the amount of GTP remaining after a GTPase reaction.

With the GTPase-Glo™ Assay, you can measure intrinsic GTPase activity, GAP-stimulated GTPase activity, GAP activity and GEF activity. The kit contains optimized reaction buffers, GTPase/GAP Buffer and GEF buffer, for performing GTPase and GAP reactions, and GEF reactions, respectively.

Easy, "Add-and-Read" Protocol

The GTPase-Glo™ Assay measures GTPase activity by detecting the amount of GTP remaining after GTP hydrolysis in a GTPase reaction. After the GTPase reaction, addition of the GTPase-Glo™ Reagent converts any remaining GTP into ATP, which is converted to a luminescent signal upon addition of the Detection Reagent.

GTPase, GAP and GEF activity is inversely correlated to the amount of light produced. A highly active GTPase hydrolyzes more GTP, reducing the amount of ATP produced from GTP and reducing light output. A less active GTPase hydrolyzes less GTP, leaving a larger amount of GTP to be converted to ATP and producing more light.

The diagram on the left shows GTPase in active and inactive phases in relation to GDP and GTP. The diagram on the right is a schematic of the GTPase-Glo™ Assay protocol.

The GTPase cycle and schematic of the GTPase-Glo™ Assay.

Monitor a Variety of GTPase-modulating Proteins (i.e., GEFS, gapS)

To mediate the process of shuttling between the active and inactive forms, GTPases need the help of two families of proteins: GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). GEFs replace the GDP bound to inactive GTPase with GTP, making the enzyme active and transducing a wide variety of cellular signals. GTPases then require the help of GAPs to hydrolyze GTP and switch off the signaling cascade.

Stimulation of GTPase Activity with GEF

In this example, Ran GTPase activity is stimulated by RCC1, a guanine nucleotide exchange factor (GEF), and detected with the GTPase-Glo™ Assay

Reactions containing 2µM of wildtype or mutant Ras (RasG12V) plus 1µM of NF1-333 (GAP protein) in GTPase/GAP Buffer.

Reactions containing 2µM of wildtype or mutant Ran (RanE70A) plus 1µM of RanGAP (GAP protein) in GTPase/GAP Buffer.

Stimulation of GTPase Activity by GAP Proteins

In this example, Ran GTPase activity is stimulated by RCC1, a guanine nucleotide exchange factor (GEF), and detected with the GTPase-Glo™ Assay

**GEF activity of RCC1.** GTPase reactions contained 2µM Ran or RanE70A, 1µM RanGAP and 1µM RCC1 in GEF Buffer. Reactions were initiated by adding 5µl of 10µM GTP in GEF Buffer containing 1mM DTT. The final reaction volume was 10µl. Reactions were incubated for 90 minutes at room temperature. To the completed GTPase reactions, 10µl of reconstituted GTPase-Glo™ Reagent was added to each well, and plates were incubated for 30 minutes at room temperature. Twenty microliters of Detection Reagent was dispensed into each reaction, plates were incubated for 5–10 minutes at room temperature and luminescence was recorded.

Sensitive Detection of GTPase-Modulating Enzymes

The GTPase-Glo™ Assay detects low nanogram amounts of GTPase-modulating enzymes in a chemical reaction.

Assessing GAP activities of NF1-333 and RanGAP with the GTPase-Glo™ Assay

This experiment shows a titration of NF1-333 or RanGAP in the presence of a fixed concentration of the cognate GTPase (Ras or Ran, respectively).

NF1-333 or RanGAP was serially diluted in GTPase/GAP Buffer, and 5µl was dispensed into each well of a 384-well plate. Five microliters of 2X GTPase-GTP solution containing 2µM Ras or 2µM Ran and 10µM GTP in GTPase/GAP Buffer was added to the dispensed GAPs. GTPase reactions were incubated for 2 hours at room temperature, then 10µl of reconstituted GTPase-Glo™ Reagent was added. After a 30-minute incubation at room temperature, 20µl of Detection Reagent was added, reactions were incubated for 5 minutes and luminescence was recorded.

Protocols

Complete Protocol

GTPase-Glo™ AssayPDF (2042 KB) – English

Specifications

Catalog Number:

What's in the box?

Item	Part #	Size	Concentration	Available Separately
rGTP	P115A	1 × 50μl	10mM	View Product
DTT	P117A	1 × 100μl	100mM	View Product
GTPase/GAP Buffer	V763A	1 × 5ml
GEF Buffer	V764A	1 × 5ml
GTPase-Glo™ Buffer	V765A	1 × 5ml
GTPase-Glo™ Reagent, 500X	V766A	1 × 15μl
Detection Reagent	V767A	1 × 10ml
ADP, 10mM	V916A	1 × 500μl

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions