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PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*


In the presence of inorganic phosphate MESG is converted to 2-amino-6-mercapto-7-methlpurine by purine nucleoside phosphorylase (EC with absorption wavelength shift to red. This feature has been used to develop our convenient MESG phosphate assay kit. Our kit provides all the essential reagents including MESG, phosphorylase and reaction buffer. The MESG substrate gives an absorbance increase at 360 nm on phosphorylysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1cm-1. The assay is shown to quantitate phosphate in solution at concentrations at least down to 2 µM. It can be used to measure the kinetics of phosphate release from phosphatases (such as GTPases and ATPases) by coupling the two enzymatic reactions.



Absorbance360 nm
Recommended plateClear UV-transparent

Absorbance microplate reader

Absorbance360 nm
Recommended plateClear bottom


Example protocol


Protocol summary

  1. Prepare 50 µL of test samples and/or phosphate standards
  2. Add 50 µL of working solution
  3. Incubate at room temperature for 30 minutes
  4. Monitor absorbance at 360 nm

Important notes
Thaw all the four components at room temperature before use.


Phosphate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21659

Add 50 μL of 1 mM KH2PO4 (Component D) into 950 μL of deionized water or enzyme reaction buffer to get 50 μM Phosphate standard solution (PS7). Take 50 μM Phosphate standard solution and perform 1:2 serial dilutions to get serially diluted Phosphate standards (PS6 - PS1) with deionized water or enzyme reaction buffer.


  1. Add 500 µL of ddH2O to the vial of MESG Substrate (Component B). Mix well by vortexing to get MESG Substrate solution. Note: 250 µl is enough for one plate.

  2. Add 100 µL of ddH2O to the vial of Purine Nucleoside Phosphorylase (PNP; Component C). Mix well by vortexing to get Purine Nucleoside Phosphorylase solution.

  3. Add the whole volume of MESG Substrate solution and Purine Nucleoside Phosphorylase solution into the bottle of Assay Buffer (Component A) and mix well to get the working solution. Place the working solution on ice. Note: This working solution is stable for at least 4 hours on ice. It is not recommended to freeze the working solution for another assay. To achieve the desirable results, UV-transparent plates or cuvettes are required. Due to the high sensitivity of this assay to Pi, it is extremely important to use Pi-free laboratory ware.


Table 1. Layout of Phosphate standards and test samples in a clear UV-transparent 96-well microplate. PS=Phosphate Standard (PS1 - PS7, 0.78 to 50 µM), BL=Blank Control, TS=Test Sample. 


Table 2. Reagent composition for each well.

PS1 - PS750 µLSerial Dilutions (0.78 to 50 µM)
BL50 µLPhosphate-free water or buffer
TS50 µLtest sample
  1. Prepare Phosphate standards (PS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of working solution to each well of Phosphate standard, blank control, and test samples to make the total assay volume of 100 µL/well. Mix the reagents thoroughly. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate at room temperature for 30 minutes.

  4. Monitor the absorbance with a microplate reader or spectrophotometer at 360 nm. Note: For cuvette assay that requires the total volume larger than 100 µL, multiply the volume of sample and assay reagent proportionally before measuring the absorption.



View all 2 citations: Citation Explorer
Sacrificial crystal templating of hyaluronic acid-based hydrogels
Authors: Thomas, Richelle C and Chung, Paul E and Modi, Shan P and Hardy, John G and Schmidt, Christine E
Journal: European Polymer Journal (2016)
Osteogenic cell cultures cannot utilize exogenous sources of synthetic polyphosphate for mineralization
Authors: Ariganello, Marianne B and Omelon, Sidney and Variola, Fabio and Wazen, Rima M and Moffatt, Pierre and Nanci, Antonio
Journal: Journal of cellular biochemistry (2014): 2089--2102


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