Malachite Green Phosphate Detection Kit #12776

#12776 Malachite Green Phosphate Detection Assay Kit Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. Malachite Green Reagent
2. Phosphate Standard (1 mM): Prepare a 0.1 mM working solution by diluting 100 µl of the Phosphate Standard provided in 900 µl dH₂O. This solution can be used to prepare a phosphate standard curve as described in the table below (see Table 1). The standard curve should provide a range between 50 and 1600 pmol per well.

B. Test Procedure

1. Creating the Standard Curve
1. Transfer 25 µl of each Phosphate Standard titration to a well of a microtiter plate.
2. Add 100 µl of Malachite Green Reagent to each well. Mix carefully without creating any bubbles. (Tip: Use a multichannel pipettor.) The experiment should be performed such that all wells will be read at approximately the same time.
3. Allow color development to proceed for 15-20 min at room temperature.
4. Measure absorbance at a wavelength between 620 and 660 nm. Subtract the absorbance of blank solution (dH₂O only) from standards.
5. Plot pmol of phosphate vs. absorbance for the standard curve.

Table 1:

Volume (µl) of diluted stock (0.1mM)	160	140	120	100	80	60	40	20	10	5	0
Volume (µl) of distilled water or diluent	90	110	130	150	170	190	210	230	240	245	250
Phosphate [pmol] per 25 µl	1600	1400	1200	1000	800	600	400	200	100	50	0



2. Enzyme Activity Assay

NOTE: Malachite Green is a highly sensitive phosphate detection solution. Make sure any reagent used in the assay is free of inorganic phosphates, as this will greatly increase the background absorbance of the assay. Enzyme reactions should be performed in a final volume of 25 µl (the same volume as that used for the standard curve). Before proceeding, confirm that your enzyme samples are free of contaminating inorganic phosphate. To determine phosphate contamination, add Malachite Green Reagent (100 µl) to 2-5 µl of sample. Any phosphate contamination will result in a color change to green. Measure absorbance at 620-660 nm and compare to standard curve to determine amount of contaminating inorganic phosphate. If required, contaminating inorganic phosphate can be removed using a desalting column or dialysis.

1. Perform phosphatase enzyme reactions in a clean plate with a minimum total reaction volume of 25 µl. Please refer to the manufacture recommended protocols for each phosphatase.
2. Transfer 25 µl of each reaction mixture to individual wells of a microtiter plate.
3. Add 100 µl of Malachite Green Reagent to each well. Mix carefully without creating any bubbles. (Tip: Use a multichannel pipettor.) The experiment should be performed such that all wells will be read at approximately the same time.
4. Allow color development to proceed for 15-20 min at room temperature.
5. Measure absorbance at a wavelength between 620 and 660 nm. Subtract the absorbance of blank solution.