Dear allen,

Attached please find publications that cover the answer to your question.

1-Biochem J. 1990 Mar 1; 266(2): 611–614.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1131175/

PMCID: PMC1131175

A continuous fluorimetric assay for ATPase activity.

U Banik and S Roy

Author information ► Copyright and License information ►

This article has been cited by other articles in PMC.

Abstract

A new continuous coupled fluorimetric assay is described for ATPases in general. Thus phosphate released from ATP hydrolysis is coupled to the nucleoside phosphorylase reaction using 7-methylguanosine as a fluorescent substrate for the nucleoside phosphorylase reaction. The hydrolysis of 7-methylguanosine leads to 7-methylguanine, which has lower quantum yield and hence can be used to monitor ATP hydrolysis continuously. The method has the potential to be extended to GTPase and nucleotidyltransferase assays

2-Phosphate Assay Kit (Fluorometric)

Rapid, simple and sensitive assay for phosphate measurement

http://www.biovision.com/phosphate-assay-kit-fluorometric-2875.html

Product Overview

Kit Summary:

• Detection method- Fluorescence (Ex/Em 535/587nm)

• Sample type- Cell and tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids, algal blooms and water from run-off areas of high fertilizer use.

• Species reactivity- Mammalian

• Applications- Measure Phosphate concentrations between 2 µM and 10 µM, with a lower detection limit of approximately 100 pmol.

Features & Benefits:

• Simple procedure; takes only ~2 hours

• Fast and convenient

• The kit can be used to detect Pi produced through reactions involving ATPases, GTPases, 5’-nucleotidase, protein phosphatases, acid and alkaline phosphatases, and phosphorylase, etc. from a variety of samples.

Kit components:

• Phosphate Assay Buffer

• PicoProbe™ (in DMSO)

• Converter

• Developer

• Phosphate Substrate

• Phosphate Standard (100 mM)

Description:

Inorganic phosphate (Pi) is one of the most important ions in biological systems. It functions in a variety of roles. One of its most important roles is as a molecular switch, turning enzyme activity on and off through the mediation of the various protein kinases and phosphatases in biological systems. A highly sensitive assay is desired to monitor Pi in a variety of samples. The newly designed and enhanced Phosphate Assay Kit provides a sensitive, easy and quick means of assessing Pi over a wide range of concentrations. In this assay, inorganic phosphate reacts with sucrose to produce glucose-1-phosphate in the presence of a proprietary enzyme. The glucose-1-phosphate is specifically oxidized to generate a product that reacts with the PicoProbe™ to generate fluorescence (Ex/Em = 535/587 nm). The kit can be used to detect Pi produced through reactions involving ATPases, GTPases, 5’-nucleotidase, protein phosphatases, acid and alkaline phosphatases, phosphorylase, etc. from a variety of samples. Unlike other commercially available assays, this assay is not affected by the presence of glucose in samples. This kit can determine phosphate concentrations between 2 µM and 10 µM, with a lower detection limit of approximately 100 pmol.

Storage Conditions:

-20°C

Shipping Conditions:

gel pack

USAGE: For Research Use Only! Not For Use in Humans.

Hoping this will be helpful,

Rafik

The nice thing about the MESG and NADH assays is that they offer the possibility of continuous measurement. If that is not required, there are many other options, including many commercial kits, although these can be expensive.

If you want to detect ADP as the product and need high sensitivity but don't want to buy expensive kits, I published a paper on a fluorescence-based method for ADP (also GDP and UDP) detection with sub-micromolar sensitivity.

http://www.ncbi.nlm.nih.gov/pubmed/21570943

This assay is suitable for miniaturization. The only reagent it requires that is not commercially available is the enzyme polynucleotide phosphorylase. The other reagents are small amounts of RiboGreen or PicoGreen or SYBR Green, and polyuridylic acid.

If you can afford it, there is a discontinuous luminescence-based assay called ADP-Glo that is very sensitive and can also be miniaturized. Although the kit is expensive, you can actually use much lower concentrations of the reagents than the manufacturer instructs, which saves a lot of money.

If you want to measure the phosphate product, and you can do a discontinuous measurement, and the ATP concentration is not too high, you can use the molybdate/Malachite Green reagent, which has sensitivity down to about 2 micromolar in an absorbance measurement.

Switching from 96-well plates to 384-well plates allows you to reduce the volumes without changing the path-length in absorbance assays. For fluorescence and luminescence assays, you can even use low-volume 384-well plates, enabling assays to be run in less than 10 µl. This assumes you have access to a suitable plate reader, of course.

You can go to the link:

http://aatbio.com/protocol/SP07042A.pdf

I hope it will be helpful.