I think the commercial kits are very similar, though you should always read the manuals for useful comparison of sensitivity, noise/signal ratio ext'.

You should also look for publications and see what is the most popular kit.

At my lab, we've been using the EnzChek® Phosphate Assay Kit (Molecular Probes®):

http://www.lifetechnologies.com/order/catalog/product/E6646

It has worked well for us, so you might want to consider it too.

You may simply use your own assay based on the work of Fiske and Subarow (1925) with some modifications by Ames and Lanzetta et al. Most of the commercially available assays are based on these articels. Using your own assay gives you the opportunity to modify it according to your needs and you save a lot of money.

Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorous. J Biol Chem 66:375–400

Ames BN (1962) Assays of inorganic phosphate, total phosphate and phosphatases.

Methods Enzymol 8:115–118

Lanzetta PA, Alvarez LJ, Reinach PS et al. (1979) An improved assay for nanomole

amounts of inorganic phosphate. Anal Biochem 100:95–97

We usually perform the assay as follows in a practical course for students to determine the ATPase activity of F- or V-type ATPases. This assay can also be adapted to microwell plates.

1. Solution A: Dissolve subsequently the following chemicals in 750 ml distilled water in a glass beaker: 4.2 g ammonium molybdate, 28.6 ml concentrated H2SO4, and 20 g sodium dodecyl sulfate (SDS). Fill up to 1 l with distilled water

Be sure that all chemicals used do not contain traces of phosphate as this will not only reduce the assay’s sensitivity but can also make the detection impossible. The phosphate reagent will turn blue immediately after preparation! To remove traces of phosphate from the glassware, all beakers, cylinders, magnetic stirrer bars, etc. were washed with methanol, followed by 1:5 diluted HCl and methanol and three times exhaustive rinsing with distilled water. We use plasticware which is also treated as described above and used only for the phosphate reagents. Never use phosphate-containing detergent for cleaning lab ware when working on phosphate determination!.

2. Solution B: 10 % (w/v) ascorbic acid. Prepare fresh before starting the experiment.

3. Calculate the needed total volume of the phosphate reagent for an experiment and prepare the reagent by mixing 6 volumes of solution A and 1 volume of solution B. Prepare fresh before the experiment.

4. Label plastic half-micro cuvettes. Perform the assay in triplicates and add cuvettes for the following blank values: buffer blank (no substrate (ATP), no protein), substrate blank (with substrate, no protein), and vesicle blank (no substrate, protein).

5. Add an appropriate volume of reaction buffer to the cuvettes. The total volume of the reaction is 300 μl. Consider the volumes of added inhibitors, vesicle fractions, substrate, etc. Add the inhibitors, e.g., 200 μM vanadate and 100 nM bafilomycin for

measuring the azide-sensitive ATP hydrolysis (F-type ATPase). Add the specific inhibitor to one-half of the cuvettes, in this example 1 mM azide, to determine the difference of ATP hydrolysis activity in the presence and absence of azide (azide-sensitive ATP hydrolysis activity. No inhibitors are necessary for total ATP hydrolysis activity.

6. Add equivalent volume of the vesicle/interphase fractions to achieve between 5 and 10 μg protein per cuvette. Mix well with the pipette tip and incubate for 30 min at RT.

7. Start the reaction with 9 μl 100 mM ATP stock solution. Mix well and incubate the reaction for 60 min.

8. Stop the reaction by addition of 700 μl phosphate reagent. Incubate for color development for 60 min and measure the absorption at 820 nm.

9. The absorption value has to be corrected for blank values to obtain the amount of liberated phosphate only by enzyme activity. The corrected absorbance value can be used to calculate phosphate concentration using a calibration curve.

Have fun!

Try perhaps the ADP-Glo from Promega.

Thank you so much for your inputs