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PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Enhanced Selectivity*

Pyrophosphate, ATP and phosphate dose responses were measured with PhosphoWorks™ Fluoremetric Pyrophosphate Assay Kit in a solid black 96-well plate using a fluorescence microplate reader.
Pyrophosphate, ATP and phosphate dose responses were measured with PhosphoWorks™ Fluoremetric Pyrophosphate Assay Kit in a solid black 96-well plate using a fluorescence microplate reader.
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Price ()
Catalog Number21614
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Pyrophosphate (PPi) is produced by a number of biochemical reactions, such as ATP hydrolysis, DNA and RNA polymerizations, cyclic AMP formation by the enzyme adenylate cyclase and the enzymatic activation of fatty acids to form their coenzyme A esters. Our PhosphoWroks™ Pyrophosphate Assay Kit provides the most robust spectrophotometric method for measuring pyrophosphate. This kit uses our proprietary fluorogenic pyrophosphate sensor that has its fluorescence intensity proportionally dependent upon the concentration of pyrophosphate. The PPi sensor used in the kit has quite high selectivity to PPi compared to phosphate and ATP. Our assay is much easier and more robust than the enzyme-coupling pyrophosphate methods that require at least two enzymes for their pyrophosphate detections. The kit provides all the essential components for assaying pyrophosphate. This kit has been successfully used in high throughput screening (HTS). Please inquire special HTS bulk package discount for the screening of >10,000 assays.

Platform


Fluorescence microplate reader

Excitation370 nm
Emission470 nm
Cutoff455 nm
Recommended plateSolid black

Components


Component A: Assay Buffer1 bottle (25 mL)
Component B: PPi Sensor1 vial (lyophilized powder)
Component C: Pyrophosphate Standard1 vial (1 mL, 50 mM)
Component D: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare PPi Sensor working solution  (50 µL)
  2. Add Pyrophosphate standards and/or test samples (50 µL)
  3. Incubate at room temperature for 10 to 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 370/470 nm (Cutoff = 455 nm)

Important notes
Thaw all the kit components 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. PPi Sensor stock solution (200X):
Add 50 µL of DMSO (Component D) into the vial of PPi Sensor (Component B) to make 200X PPi Sensor stock solution. Note: 25 µL of the PPi Sensor stock solution is enough for one 96-well plate.

 

PREPARATION OF STANDARD SOLUTION

Pyrophosphate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/21614

Prepare 1 mM Pyrophosphate standard by adding 10 µL of 50 mM Pyrophosphate Standard (Component C) into 490 µL of Assay Buffer (Component A), or buffer of your choice (preferably 50 mM Hepes buffer, pH 7) to make 1 mM Pyrophosphate standard. Take 1 mM Pyrophosphate standard (PS7) to perform 1:3 serial dilutions to get serially diluted Pyrophosphate standards (PS6-PS1) with Assay Buffer (Component A).

PREPARATION OF WORKING SOLUTION

 Add 25 µL of 200X PPi Sensor stock solution to 5 mL of Assay Buffer (Component A), and mix them well. Note: Due to the high sensitivity of this assay to PPi, it is important to use PPi-free labware and reagents. Note: DTT (1 uM) will increase the background, MgCl2 ≥ 2mM decrease the response.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of pyrophosphate standards and test samples in a solid black 96-well microplate.  PS = Pyrophosphate Standard, 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 Dilution (1 to 1000 µM)
BL 50 µL Assay Buffer (Component A)
TS 50 µL Sample
  1. Prepare Pyrophosphate 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 PPi Sensor working solution to each well of Pyrophosphate standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of  PPi Sensor working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 370/470 nm (Cutoff = 455 nm)

Citations


View all 7 citations: Citation Explorer
Biological studies and target engagement of the 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD)-targeting antimalarial agent (1 R, 3 S)-MMV008138 and analogs
Authors: Ghavami, Maryam and Merino, Emilio F and Yao, Zhong-Ke and Elahi, Rubayet and Simpson, Morgan E and Fern{\'a}ndez-Murga, Maria L and Butler, Joshua H and Casasanta, Michael A and Krai, Priscilla M and Totrov, Maxim M and others,
Journal: ACS infectious diseases (2017): 549--559
Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B
Authors: King, Dustin T and Wasney, Gregory A and Nosella, Michael and Fong, Anita and Strynadka, Natalie CJ
Journal: Journal of Biological Chemistry (2017): 979--993
Biological studies and target-engagement of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD)-targeting antimalarial agent (1R, 3S)-MMV008138 and analogs
Authors: Ghavami, Maryam and Merino, Emilio Fern and o , undefined and Yao, ZhongKe and Elahi, Rubayet and Simpson, Morgan and Fern, undefined and ez-Murga, Maria and Butler, Joshua Hayden and Casasanta, Michael and Krai, Priscilla and Totrov, Maxim and others, undefined
Journal: ACS Infectious Diseases (2017)
Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae
Authors: Sabra, Ayman and Bessoule, Jean-Jacques and Atanasova-Penichon, Vessela and No{\"e}l, Thierry and Dementhon, Karine
Journal: Infection and immunity (2014): 413--422
Biomarkers identified by urinary metabonomics for noninvasive diagnosis of nutritional rickets
Authors: Wang, Maoqing and Yang, Xue and Ren, Lihong and Li, Songtao and He, Xuan and Wu, Xiaoyan and Liu, Tingting and Lin, Liqun and Li, Ying and Sun, Changhao
Journal: Journal of proteome research (2014): 4131--4142
Host-Pathogen Interaction and Signaling Molecule Secretion Are Modified in the dpp3 Knockout Mutant of Candida lusitaniae
Authors: Sabra, Ayman and Bessoule, Jean-Jacques and Atanasova-Penichon, Vessela and Noel, Thierry and Dementhon, Karine
Journal: Infection and immunity (2014): 413--422