Figure 1. A linear standard curve was generated using phosphate standards and the Malachite Green Phosphate Detection Kit. The phosphate standard was diluted in dH2O as described in the protocol section and samples were assayed with the Malachite Green Phosphate Detection Kit. This standard curve is for demonstration purposes only; users should generate a standard curve for each sample set in order to accurately determine phosphate concentration.
Figure 2. Receptor type protein phosphatase F (PTPRF) activity was measured using the Malachite Green Phosphate Detection Kit. PTPRF phosphatase activity was monitored using tyrosine phosphopeptide-2 (DADE(pY)LIPQQG) as the substrate. The reactions were performed with various amounts of PTPRF phosphatase at 37ºC for 10 minutes in a buffer containing 50 mM imidazole, 0.2% 2-mercaptoethanol, 65 ng/μl BSA, and 1 mM tyrosine phosphopeptide-2 (DADE(pY)LIPQQG).
|Product Includes||Quantity (with Count)||Storage Temp|
|Malachite Green Reagent||2 x 28 ml||RT|
|Phosphate Standard (1 mM)||1 x 0.5 ml||RT|
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
|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|
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.
posted June 2020
Protocol Id: 2058
The Malachite Green Phosphate Detection Kit is a convenient and sensitive, single-step free-phosphate determination kit that can be used for measuring phosphate released during enzymatic phosphatase assays.
The Malachite Green Phosphate Detection Kit detects only free-phosphate in sample. Figure 1 demonstrates a linear range of 50 to 1600 pmol of phosphate.
The Malachite Green Phosphate Detection Kit is designed for the in vitro measurement of phosphatase activity. Protein phosphatase enzymatic activity results in the release into solution of free inorganic phosphate from protein-phosphatase substrates. This free inorganic phosphate then forms a green molybdophosphoric acid complex. Formation of this complex is monitored and quantified by measuring the absorbance at 630 nm. This absorbance is proportional to free-inorganic phosphate in solution and correlates with the activity of phosphatase (1,2).