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12776
Malachite Green Phosphate Detection Kit
Cellular Assay Kits
Assay Kit

Malachite Green Phosphate Detection Kit #12776

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Malachite Green Phosphate Detection Kit: Image 1

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.

Malachite Green Phosphate Detection Kit: Image 2

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).

To Purchase # 12776S
Product # Size Price
12776S
1 Kit  (500 assays) $ 144

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

Protocol

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#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
  3. 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.

posted June 2020

Protocol Id: 2058

Product Description

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.

Specificity / Sensitivity

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.

Background

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).

  1. Baykov, A.A. et al. (1988) Anal Biochem 171, 266-70.
  2. Geladopoulos, T.P. et al. (1991) Anal Biochem 192, 112-6.

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