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 2.4.2.1) 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.

Example protocol

AT A GLANCE

Protocol summary

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

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

PREPARATION OF STANDARD SOLUTION

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.

PREPARATION OF WORKING SOLUTION

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.
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.
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.

SAMPLE EXPERIMENTAL PROTOCOL

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.

BL	BL	TS	TS
PS1	PS1	...	...
PS2	PS2	...	...
PS3	PS3
PS4	PS4
PS5	PS5
PS6	PS6
PS7	PS7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
PS1 - PS7	50 µL	Serial Dilutions (0.78 to 50 µM)
BL	50 µL	Phosphate-free water or buffer
TS	50 µL	test sample

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.
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.
Incubate at room temperature for 30 minutes.
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.

Citations

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

References

View all 8 references: Citation Explorer

Relationship between activating and editing functions of the adenylation domain of apo-tyrocidin synthetase 1 (apo-TY1)
Authors: Bucevic-Popovic V, Pavela-Vrancic M, Dieckmann R, Von Dohren H.
Journal: Biochimie (2006): 265

Purine nucleoside phosphorylase activity in rat cerebrospinal fluid
Authors: Silva RG, Santos DS, Basso LA, Oses JP, Wofchuk S, Portela LV, Souza DO.
Journal: Neurochem Res (2004): 1831

Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors
Authors: Tan YC, Wu H, Wang WN, Zheng Y, Wang ZX.
Journal: Anal Biochem (2002): 156

A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases
Authors: Rieger CE, Lee J, Turnbull JL.
Journal: Anal Biochem (1997): 86

Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay
Authors: Melki R, Fievez S, Carlier MF.
Journal: Biochemistry (1996): 12038

Page updated on December 17, 2024

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