Pyrophosphate Assay Kit (Fluorometric) (ab112155)

Reviews (1)Q&A (29)References (4)
Urinary total pyrophosphate (PPi) concentrations were determined using ab112155
  • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
  • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
  • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
  • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
  • Functional Studies - Pyrophosphate Assay Kit (Fluorometric) (ab112155)

Key features and details

  • Assay type: Quantitative
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Sample type: Adherent cells, Cell culture extracts, Plasma, Serum, Suspension cells, Tissue Lysate, Urine

Overview

  • Product name

    Pyrophosphate Assay Kit (Fluorometric)
    See all Pyrophosphate kits

  • Detection method

    Fluorescent

  • Sample type

    Urine, Serum, Plasma, Cell culture extracts, Adherent cells, Suspension cells, Tissue Lysate

  • Assay type

    Quantitative

  • Product overview

    Pyrophosphate Assay Kit (Fluorometric) ab112155 provides the most robust spectrophotometric method for the measurement of pyrophosphate.


    The pyrophosphate assay protocol uses a proprietary fluorogenic pyrophosphate sensor that has its fluorescence intensity proportionally dependent upon the concentration of pyrophosphate.


    This pyrophosphate assay protocol is much easier and more robust than enzyme-coupling pyrophosphate methods, which require at least two enzymes for their pyrophosphate detections. Due to its direct measurement of pyrophosphate, this kit is ideal for screening inhibition or activities of enzymes that consume or generate pyrophosphate.


    The pyrophosphate assay is an optimized mix-and-read assay and can be performed in a convenient 96-well or 384-well microtiter-plate format.


    Assay time: 10-30 minutes

  • Notes

    This product measures PPi formation at Ex/Em = 316/456 nm.

    If your instrument has filters for Ex/Em = 370/470 nm, we recommend using Pyrophosphate Assay Kit II (Fluorometric) (ab179836).

    Pyrophosphates (PPi) are 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.

  • Platform

    Microplate reader

Properties

Associated products

    Images

    • Urinary total pyrophosphate (PPi) concentrations were determined using ab112155
      Urinary total pyrophosphate (PPi) concentrations were determined using ab112155Caballero D et al., PLoS One, 12(7). Fig 1a. doi: 10.1371/journal.pone.0180098 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

      Urine PPi was corrected for urine creatinine, which was measured by LC-MS/MS or by ELISA using appropriate controls to adjust for inter-assay variability.

    • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)

      Pyrophospfate measured in cell lysates showing quantity (nmol) per 106cells

    • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)

      Pyrophosphate measured in tissue lysates showing quantity (nmol) per microgram protein

    • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)

      Pyrophosphate measured in biofluids showing concentration (micromolar). Obtained values were recalculated from 1:1000 sample dilutions.

    • Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Functional Studies- Pyrophosphate Assay Kit (Fluorometric) (ab112155)

      Example PPi standard curve. 

      Data provided for demonstration purposes only.

    • Functional Studies - Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Functional Studies - Pyrophosphate Assay Kit (Fluorometric) (ab112155)
      Pyrophosphate, ATP and phosphate dose responses were measured with ab112155 in a solid black 96-well plate using a fluorescence microplate reader. As low as 1 µM (100 picomoles/well) pyrophosphate can be detected with 10 minutes incubation.

    References (4)

    Publishing research using ab112155? Please let us know so that we can cite the reference in this datasheet.

    ab112155 has been referenced in 4 publications.

    • Caballero D  et al. Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice. PLoS One 12:e0180098 (2017). PubMed: 28704395
    • Ao M  et al. Overlapping functions of bone sialoprotein and pyrophosphate regulators in directing cementogenesis. Bone 105:134-147 (2017). PubMed: 28866368
    • Zhang F  et al. The cAMP phosphodiesterase Prune localizes to the mitochondrial matrix and promotes mtDNA replication by stabilizing TFAM. EMBO Rep 16:520-7 (2015). Functional Studies . PubMed: 25648146
    • Luna E  et al. Plant perception of ß-aminobutyric acid is mediated by an aspartyl-tRNA synthetase. Nat Chem Biol 10:450-6 (2014). PubMed: 24776930

    Customer reviews and Q&As

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    1-10 of 30 Abreviews or Q&A

    Question

    Hi, I have some technical questions about the product 
    Pyrophosphate Assay Kit II (Fluorometric) (ab179836)
     

     

    1) The closest wavelength on my instrument is Ex/Em=355/460 rather than the recommended Ex/Em=370/470 of this product. Do you think I can still use this product to perform my experiments? 

     

    2) Another Pyrophosphate Assay Kit ab112155 uses the wavelength of Ex/Em = 316/456, and it's $80 cheaper than ab179836, do you think I can use either of them or is anyone outperform than the other? 

     

    3) My samples are reaction mixture rather than the described type of tissue or cell extract. Is this product suitable for my experiment? 

     

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    Answer

    Thank you for contacting us.

    To answer your questions:

    1) Yes you can use ab179836 with the settings on your instruments

    2) Ab112155 would not work for you, excitation 355 is too high for this kit

    3) Yes, your samples will work with this kit


    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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    Question

    Thank you very much for your standard curve. It's very helpful.

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    Answer

    Thank you for your kind response. It is a pleasure to help in any way I can. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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    Measuring PPi in mouse serum

     Average Average 3/5 (Ease of Use)
    Abreviews
    abreview image
    We wanted to measure PPi in mouse blood. A big problem is the high autofluorescence of serum and plasma, possibly contributed by albumin. Therefore we tested several conditions:
    - untreated serum
    - heat-inactivated serum (45 min 55°C)
    - plasma
    - serum after centrifugation through a 300 kDa cut-off-membrane
    PPi amount was expected in a range between 0.5 - 5 µM [Koba et al. 2005]. Only the last condition (cut-off-membrane) resulted in the expected measurement (approximately 1.62 µM).

    Abcam user community

    Verified customer

    Submitted Jan 19 2017

    Question

    RIPA buffer interfere with assay?

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    Answer

    I can confirm that SDS, Tween-20, and EDTA (> 30 um) will interfere with the assay. Since RIPA buffer has SDS in it, it is not suitable.

    TritonX-100 up to 0.5% does not interefere.


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    Question

    Pyrophosphate Assay Kit questions

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    Answer

    1. What are the maximum and minimum limits of detection?

    The detection limit is ˜0.3 - 1 uM and the Max is 10 uM for ab112155, and 1 mM for ab179836.

    2. Are the assays compatible with citrated plasma (plasma collected with citrate)?

    Citrated plasma will not be a problem, but adjust to pH 7 with Hepes buffer if possible.

    3. Is there any cross-reaction with phosphates other than pyrophosphate? My samples are mouse plasma and urine.

    No.

    4. What are the advantages of the two assays, 21614 vs.21611? Why would I choose one over the other?

    Ab112155 has a slightly lower detection limit (˜0.3 uM), but more ATP interference. Ab179836 has better linearity, and can detect as high as 1 mM PPI.

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    Question

    We use a buffer containing the following with our samples. Will we be able to use this kit, or will the buffer interfere?

    50mM HEPES pH 8, 150mM NaCl, 1mM DDT, 2mM MgCl2

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    Answer

    The components of this buffer should not interfere with this assay.

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    Question

    my samples have 3 mM dATP, will this affect the sensitivity of the assay?

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    Answer

    Thank you for contacting us. I did contact the lab regarding whether ATP or dATP in samples will compromise the sensitivity of the assay and they did not think it would have much effect. Please let me know if you have any further questions.

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    Question

    For the pyrophosphate assay kit, is it possible to use the kit above room temperature? What is the maximum temperature the kit has been found to work? Thanks.

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    Answer

    37C should be no problem. We did not test with a higher temperature, however, 50C might be OK in theory.

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    Question

    I see the probe used in this kit is proprietary in nature but I was wondering if any sturctural information is available for it?

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    Answer

    It is a proprietary dye so unfortunately we can't disclose the structure.

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    Question

    preparation of yeast samples? cell lysis?

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    Answer



    J'ai reçu la confirmation qu'un tampon de lyse standard pour les levures peut être utilisé avec le kit PPi.
    Le laboratoire où a été développé ce kit n'a malheureusement pas de données expérimentales pour les levures.

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    1-10 of 30 Abreviews or Q&A

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