PhosphoWorks™ Fluorimetric Phosphate Assay Kit Red Fluorescence

Cells utilize a wide variety of phosphate (Pi) and polyphosphate esters as enzyme substrates, second messengers, membrane structural components and vital energy reservoirs. Phosphate is involved in many biological processes. For example, phosphatases, ATPases and several other enzymes catalyze biochemical reactions in which inorganic phosphate is released from a phosphoester substrate. Detection of many phosphoester-metabolizing enzymes is difficult because suitable substrates are not available. It usually has been necessary to determine inorganic phosphate release using tedious colorimetric assays or radioisotope-based methods. This PhosphoWorks™ Fluorimetric Phosphate Assay Kit has been developed for measuring the activity of any Pi-generating enzyme using our red fluorescent phosphate sensor. The kit provides sensitive detection of Pi, an alternative to hazardous radioactive methods and other less sensitive colorimetric assays. The measurement of Pi is based on the change in the absorbance and fluorescence of our new phosphate sensor. Our kit provides all the essential reagents including phosphate sensor, phosphate standards and assay buffer. The assay is shown to quantitate phosphate in solution at concentrations at least down to 0.1 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. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation steps required.

Example protocol

AT A GLANCE

Protocol summary

Prepare test samples or Phosphate standards(40 µL)
Add equal volume of Assay Buffer (40 µL)
Add Phosphate Sensor working solution (20 µL)
Incubate at room temperature for 15 to 60 minutes
Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw one of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Phosphate Sensor stock solution (125X):
Add 20 µL of DMSO into Phosphate Sensor (Component B) to make 125X Phosphate Sensor stock solution.

PREPARATION OF STANDARD SOLUTION

Phosphate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21660

Add 50 µL of 1 mM KH₂PO₄ (Component C) into 950 µL of deionized water or enzyme reaction buffer to get a 50 µM Phosphate standard solution (PS7). Take 50 µM Phosphate standard solution (PS7) and perform 1:2 serial dilutions to get serially diluted Phosphate standards (PS6 - PS1) with deionized H₂O or enzyme reaction buffer.

PREPARATION OF WORKING SOLUTION

Add 20 μL of 125X Phosphate Sensor stock solution into 2.5 mL of sterile H₂O and mix well to make Phosphate Sensor working solution. Avoid potential Pi contamination. Note: Avoid direct exposure of Phosphate Sensor (Component B) to light. Due to the high sensitivity of this assay to Pi, it is extremely important to use Pi-free laboratory ware and reagents.

SAMPLE EXPERIMENTAL PROTOCOL

Run the phosphate assay at pH 6.5 to 7.4

Table 1. Layout of Phosphate standards and test samples in a solid black 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	40 µL	Serial Dilutions (0.78 to 50 µM)
BL	40 µL	H₂O or Enzyme Reaction Buffer
TS	40 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 20 µL of reagent per well instead of 40 µL.
Add 40 µL of Assay Buffer (Component A) and 20 µL of Phosphate Sensor working solution to each well of Phosphate standard, blank control, and test samples to make the total Phosphate assay volume of 100 µL/well. For a 384-well plate, add 20 µL of Assay Buffer (Component A) and 10 µL of Phosphate Sensor working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction mixture at room temperature for 15 to 60 minutes.
Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).

Citations

View all 6 citations: Citation Explorer

Pseudomonas aeruginosa Phosphate Transporter PitA (PA4292) Controls Susceptibility to Aminoglycoside Antibiotics by Regulating the Proton Motive Force
Authors: Zhao, Xinrui and Jin, Yongxin and Bai, Fang and Cheng, Zhihui and Wu, Weihui and Pan, Xiaolei
Journal: Antimicrobial Agents and Chemotherapy (2022): e00992--22

Bladder epithelial cell phosphate transporter inhibition protects mice against uropathogenic Escherichia coli infection
Authors: Pang, Yu and Cheng, Zhihui and Zhang, Si and Li, Shujie and Li, Xueping and Li, Xiaodan and Zhang, Xiao and Li, Xiaoxiao and Feng, Yingxing and Cui, Heting and others,
Journal: Cell Reports (2022): 110698

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 (2017): 487--496

It's a NO for Bacterial Settlement: Nitric Oxide Regulated Biofilm Formation and Protein Expression
Authors: Xu, Yueming
Journal: (2014)

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 November 23, 2024

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