PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Enhanced Selectivity* |
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 316 nm |
Emission | 456 nm |
Cutoff | 420 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
- Prepare Pyrophosphate standards and/or test samples (50 µL)
- Add Pyrophosphate working solution (50 µL)
- Incubate at room temperature for 10 to 30 minutes
- Monitor fluorescence intensity at Ex/Em = 316/456 nm (Cutoff = 420 nm)
Thaw all the four components at room temperature before use.
PREPARATION OF STOCK SOLUTIONS
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
Add 50 µL of DMSO (Component D) into the vial of PPi Sensor (Component B) to make 200X PPi Sensor stock solution. Protect from light.
Note 25 µL of the PPi Sensor Stock Solution is enough for one 96-well plate.
Add 10 µL of 50 mM Pyrophosphate Standard (Component C) into 490 µL of ddH2O or 50 mM Hepes buffer (pH 7) to make 1 mM Pyrophosphate standard solution.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/21611
PREPARATION OF WORKING SOLUTION
Add 25 μL of 200X PPi Sensor stock solution to 5 mL of Assay Buffer (Component A) and mix well to make PPi working solution.
Note Due to the high sensitivity of this assay to PPi, it is important to use PPi-free labware and reagents. DTT ≥ 1 mM will increase the background, MgCl2 ≥ 2 mM will 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 (PS1 - PS7, 0.14 to 100 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 (0.14 to 100 µM) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | test sample |
- 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.
- Add 50 µL of PPi working solution to each well of Pyrophosphate standard, blank control, and test samples to make the total Pyrophosphate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of PPi working solution into each well instead, for a total volume of 50 µL/well. Mix the reagents thoroughly.
- Incubate at room temperature for 10 to 30 minutes.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 316/456 nm (Cutoff = 420 nm).
Images
Citations
Authors: Sun, Fan and Cao, Xueqiang and Yu, Dianzhen and Hu, Dongqiang and Yan, Zheng and Fan, Yingying and Wang, Cheng and Wu, Aibo
Journal: Molecular Plant-Microbe Interactions (2022): 416--427
Authors: Rattazzi, Marcello and Faggin, Elisabetta and Bertacco, Elisa and Buso, Roberta and Puato, Massimo and Plebani, Mario and Zaninotto, Martina and Condotta, Davide and Zoppellaro, Giacomo and Pagliani, Leopoldo and others, undefined
Journal: Journal of Cardiovascular Translational Research (2018): 1--10
Authors: Andrukhova, Olena and Sch{\"u}ler, Christiane and Bergow, Claudia and Petric, Alexandra and Erben, Reinhold G
Journal: Frontiers in endocrinology (2018): 311
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
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
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)
Authors: Xu, Yueming
Journal: (2014)
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
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
Authors: Sabra, Ayman and Bessoule, Jean-Jacques and Atanasova-Penichon, Vessela and Noel, Thierry and Dementhon, Karine
Journal: Infection and immunity (2014): 413--422
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Application notes
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