PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Alternative formats
PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Enhanced Selectivity* |
Related products
Overview | ![]() ![]() |
See also: Enzymes, Polymerases
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. 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
Excitation | 316 nm |
Emission | 456 nm |
Cutoff | 420 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol Summary
- 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)
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.
Note 25 µL of the PPi Sensor Stock Solution is enough for one 96-well plate.
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. Protect from light.Note 25 µL of the PPi Sensor Stock Solution is enough for one 96-well plate.
2. Pyrophosphate standard solution (1 mM)
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 SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/21611
https://www.aatbio.com/tools/serial-dilution/21611
Pyrophosphate standard
Add 50 μL of 1 mM Pyrophosphate standard solution into 450 μL of ddH2O or 50 mM Hepes buffer to get 100 μM Pyrophosphate standard solution (PS7). Take 100 μM Pyrophosphate standard solution and perform 1:3 serial dilutions in ddH2O or 50 mM Hepes buffer to get serially diluted Pyrophosphate standards (PS6 - PS1).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.
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, 0.13 to 100 µM), BL = Blank Control, TS = Test Sample.
Table 2. Reagent composition for each well.
BL | BL | TS | TS |
PS1 | PS1 | ... | ... |
PS2 | PS2 | ... | ... |
PS3 | PS3 | ||
PS4 | PS4 | ||
PS5 | PS5 | ||
PS6 | PS6 | ||
PS7 | PS7 |
Well | Volume | Reagent |
PS1 - PS7 | 50 µL | Serial Dilutions (0.13 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
View all 11 citations: Citation Explorer
AaTAS1 and AaMFS1 Genes for Biosynthesis or Efflux Transport of Tenuazonic Acid and Pathogenicity of Alternaria alternata
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: 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
RANKL Expression Is Increased in Circulating Mononuclear Cells of Patients with Calcific Aortic Stenosis
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: 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
Augmented fibroblast growth factor-23 secretion in bone locally contributes to impaired bone mineralization in chronic kidney disease in mice
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: Andrukhova, Olena and Sch{\"u}ler, Christiane and Bergow, Claudia and Petric, Alexandra and Erben, Reinhold G
Journal: Frontiers in endocrinology (2018): 311
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
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
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)
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)
It's a NO for Bacterial Settlement: Nitric Oxide Regulated Biofilm Formation and Protein Expression
Authors: Xu, Yueming
Journal: (2014)
Authors: Xu, Yueming
Journal: (2014)
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
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
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
Authors: Sabra, Ayman and Bessoule, Jean-Jacques and Atanasova-Penichon, Vessela and Noel, Thierry and Dementhon, Karine
Journal: Infection and immunity (2014): 413--422
References
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Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs
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Authors: Campbell K, Swann K.
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An efficient method for quantitative determination of cellular ATP synthetic activity
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Authors: Hara KY, Mori H.
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Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium leprae by bioluminescence assay (ATP method)
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Authors: Yamazaki T, Gidoh M, Matsuoka M.
Journal: Nihon Hansenbyo Gakkai Zasshi (2006): 227
Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase
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Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay
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Application notes
Selective Detection of Pyrophosphate Using a Fluorogenic Pyrophosphate Sensor
Abbreviation of Common Chemical Compounds Related to Peptides
β-adrenoceptors are upregulated in human melanoma and their activation releases pro-tumorigenic cytokines and metalloproteases in melanoma cell lines
Bio-inspired NADH regeneration by carbon nitride photocatalysis using diatom templates
Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila
Abbreviation of Common Chemical Compounds Related to Peptides
β-adrenoceptors are upregulated in human melanoma and their activation releases pro-tumorigenic cytokines and metalloproteases in melanoma cell lines
Bio-inspired NADH regeneration by carbon nitride photocatalysis using diatom templates
Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila