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Malachite Green Assay for Inorganic Phosphate
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
1. Mix an equal volume (50 to 100 μl) of a nucleotide hydrolysis reaction with an equal volume of the Malachite Green solution in a flat-bottomed microtiter dish (see Hint #1).

2. Set up an 8 point standard curve of 0.1 to 1.0 nmols of Potassium Phosphate,(monobasic) in duplicate each time the assay is done. Also set up negative control reactions (e.g. no protein) in the same buffer as the experimental samples (see Hint #2).

3. Read Absorbance at 630 or 650 nm, 10 to 20 min after adding dye (be consistent). It's easiest if you have a microtiter plate reader. If not, you can set up the reactions in individual tubes in a larger volume, and measure Absorbance's separately in a spectrophotometer with plastic curettes (see Hint #3 and Hint #4).

Nucleotides   Use a high quality ATP
Malachite Green Solution   Bring to 1 liter total volume with 0.7 M HCl. Store in dark bottle at 4°C.
0.3 g Malachite Green Oxalate
2 g Sodium Molybdate
0.5 g Triton X-100
BioReagents and Chemicals
Potassium Phosphate, Monobasic
Triton X-100
Hydrochloric Acid
Malachite Green Oxalate
Sodium Molybdate
Protocol Hints
1. The Perchloric Acid stop step mentioned in the J. Biochem. paper is not necessary unless your NTPase works in 0.7 M HCl.

2. It is especially critical to have the same amounts of nucleotides and glycerol, as these affect the background absorbance.

3. This assay works very well to detect 0.1 to 1.0 nmol of liberated inoragnic phosphate during nucleotide hydrolysis experiments. The limitations of this method are that the nucleotide concentration should be kept to less than 300 μM, and certain substances (notably glycerol) inhibit the reaction. To detect smaller amounts of phosphate (10 to 60 nmol) use a different procedure (P. Gonzalez-Romo, et al.), since this assay can tolerate up to 30 mM ATP.

4. The less nucleotide you can have in the reaction the better, since nucleotides give a background absorbance. For ATP, a concentration of 300 μM works well; GTP gives a higher background, so less should be used if you can get away with it. You generally want to work at least at ten times the rate constant (Km), but you might be able to use less nucleotide if hydrolysis is linear for a long time. The color development is inhibited by glycerol at greater than 2 % final concentration or by citrate.

Citation and/or Web Resources
1. Kodama S, Tamaki N, Mukai T, Yonekura Y, Torizuka K, Suzuki Y, Tamaki S, Nohara R, Kambara H, Kawai C. [Asynchronous wall motion in patients with ischemic heart disease assessed by higher-order harmonics of the Fourier series]. J Cardiogr Suppl. 1986;8:25-32. Japanese.
2. Gonzalez-Romo P, Sanchez-Nieto S, Gavilanes-Ruiz M. A modified colorimetric method for the determination of orthophosphate in the presence of high ATP concentrations. Anal Biochem. 1992 Feb 1;200(2):235-238.