Overview

  • Product name

    Pyrophosphate Assay Kit II (Fluorometric)
    See all Pyrophosphate kits
  • Detection method

    Fluorescent
  • Sample type

    Serum, Plasma, Cell Lysate, Tissue Lysate
  • Assay type

    Quantitative
  • Sensitivity

    >= 1 µM
  • Assay time

    0h 10m
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    Pyrophosphate (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.

    Pyrophosphate Assay Kit II (Fluorometric) (ab179836) provides the most robust spectrophotometric method for the measurement of pyrophosphate. It 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 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 with enhanced selectivity to pyrophosphate. The assay is an optimized mix-and-read assay and can be performed in a convenient 96-well or 384-well microtiter-plate format. The kit provides all the essential components for assaying pyrophosphate.

  • Notes

    This product measures PPi formation at Ex/Em = 370/470 nm.

    If your instrument has filters for Ex/Em = 316/456 nm, we recommend using Pyrophosphate Assay Kit (Fluorometric) (ab112155).

  • Platform

    Microplate reader

Properties

Images

  • Pyrophosphate measured in mouse tissue lysates showing quantities (nanomoles) per microgram of protein tested. The lysates were tested in the range of 2-50 microgram of proteins per mL, data shown from duplicates, +/- SD.

  • Pyrophosphate measured in cell lysates showing quantity (micromoles) per 1 million cells tested.

  • Pyrophosphate measured in biological fluids showing concentration (millimolar). The samples were tested in the dilution range of 1:200-1:5000.

  • Pyrophosphate, ATP and phosphate dose responses were measured with the Abcam Pyrophosphate Assay Kit II (Fluorometric) (ab179836) in a solid black 96-well plate using a fluorescence microplate reader. As low as 1 µM (100 picmoles/well) pyrophosphate can be detected with 10 minutes incubation.

Protocols

References

This product has been referenced in:

  • Xiong L  et al. Osteoblastic Lrp4 promotes osteoclastogenesis by regulating ATP release and adenosine-A2AR signaling. J Cell Biol 216:761-778 (2017). Read more (PubMed: 28193701) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
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.

Read More
Abreviews
Abreview for ab179836 - Pyrophosphate Assay Kit II (Fluorometric).
We are working with the vacuolar H+-pyrophosphatase AVP1 (AT1G15690) in Arabodopsis thaliana. AVP1 is a transmembrane protein, allocated principally in vacuolar membranes (Gaxiola et. al 2012). This enzyme has a PPiase activity, and couple the energy release from the PPi to transport H+ against the gradient (from cytoplasm to vacuole), energizing the vacuolar lumen. Based in phenotypic observations of AVP1 knock-out plants, as well as in published literature (Marsh et al. 2000), our hypothesis suggests that under a steep pH gradient this protein is able to synthesize PPi. In tonoplast there is another transmembrane protein, called vacuolar ATPase, capable to generate a steep pH gradient between cytoplasm and vacuole, using ATP hydrolysis as a motive force.
To prove our hypothesis we isolate tonoplast from A. thaliana seedlings, using a sucrose gradient, and we test PPi synthesis activity in these samples using Pyrophosphate Assay Kit II (ab179836).
In the PPi synthase reaction we have: 1. ATP and Cl2Mg (4 mM and 5 mM respectively) to feed the vacuolar ATPase and to generate a steep pH gradient between cytoplasm and vacuole 2. K2HPO4 (2.5 mM) as a Pi source in PPi synthesis reaction 3. KCl (50 mM) since AVP1 is K+ dependent 4. Sorbitol (250 mM) as a membrane stabilizer 5. NaF (10 mM) as a PPiase activity inhibitor 6. Buffer HEPES (pH 7.2 – 100 mM) and 7. Tonoplast sample (diluted 10 times). The reaction time lasts 60 minutes at 30 ºC and then we boil the samples for 5 minutes. We measure PPi concentration at the initial and final point.
Our equipment “Fluoroskan Ascent FL” has an excitation filter at 355 nm and an emission filter at 485 nm. According with the PPi sensor excitation/emission spectra, seen in the figure 2 in the ab179836 manual, we have (with our filters) almost 100% of the PPi sensor excitation and about 80% of the PPi sensor emission.
In our first approach to the Pyrophosphate Assay Kit II (ab179836) we can see:
1. We performed a PPi dose response curve (same as in figure 1 in the ab179836 manual) with the standard provided by the KIT and we obtained a lower RFU values. This is a problem because we can not use this curve to calculate PPi concentration in our testing samples, being that they have higher RFU values (figure 1A). Then, we made another PPi dose response curve with a home-made PPi standard. We saw normal RFU values (20 to 200) in PPi concentration ranging between 1 to 100 mM. This result suggests that in our hands the Pyrophosphate Assay Kit II has relatively low sensitivity (figure 1B).
2. We tested the PPi sensor background (without PPi) and the RFU values are really low (1.80 ± 0.33) comparing with water samples (1.27 ± 0.038). By contrast, the PPi synthase reaction buffer showed relatively high background (RFU 9.58 ± 2.09). In the other hand, our tonoplast isolation protocol produces samples with some quantity of chlorophyll. We saw that this molecule has autofluorescens (RFU 98.79 ± 19.1), but we can avoid chlorophyll autofluorescens boiling the samples prior to the PPi quantification (RFU 2.72 ± 0.771). (Figure 2)
3. Finally we tested PPi synthesis activity in two different tonoplast samples (Col vs mutant1). We can see an increase in PPi levels in both samples after 60 minutes of PPi synthesis reaction (Col 17.72 vs mutant1 6.06 PPi μmol * μg protein-1 * hs-1 respectivelly - Figure 3).

Dr. Gaston Pizzio

Verified customer

Submitted May 19 2014

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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