Overview

  • Product name

    ADP Assay Kit (Colorimetric/Fluorometric)
    See all ADP kits
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

    Urine, Serum, Plasma, Cell culture extracts, Other biological fluids, Tissue Extracts, Cell Lysate, Cell culture media
  • Assay type

    Quantitative
  • Sensitivity

    > 1 µM
  • Assay time

    1h 00m
  • Species reactivity

    Reacts with: Mammals, Other species
  • Product overview

    ADP Assay Kit (Colorimetric/Fluorometric) (ab83359) provides a convenient colorimetric and fluorometric method to measure ADP level.


    In the ADP assay, ADP is converted to ATP and pyruvate. The generated pyruvate can be quantified by colorimetric (ODmax = 570 nm) or fluorometric method (Ex/Em 535/587 nm).


    Conventionally, ADP levels are measured by luciferase/luciferin mediated assays after ADP is converted to ATP. However, the luciferase system is unstable and luminescence equipment is not generally available in most laboratories. In comparison, this assay is simple, sensitive, stable and high-throughput adaptable and can be used with conventional microplate readers.


    The ADP assay can detect as low as 1 μM ADP in biological samples.

  • Notes

    ADP is a product of ATP de-phosphorylation and it can be rephosphorylated to ATP. De-phosphorylation and re-phosphorylation occur via various phosphatases, phosphorylases and kinases. ADP is stored in platelets and can be released to interact with a variety of purinergic receptors. ADP levels regulate several enzymes involved in intermediary metabolism. ADP conversion to ATP primarily occurs within the mitochondrion and chloroplast although several such processes occur in the cytoplasm.

  • Platform

    Microplate reader

Properties

Images

  • ADP levels measured fluorometrically in cell culture supernatants (background signal subtracted, mean of duplicates; +/- SD).

  • ADP levels measured fluorometrically in cell lysates (background signal subtracted, mean of duplicates; +/- SD).

  • APD levels colorimetrically measured in biological fluids (rat plasma and serum, human saliva); background signal subtracted, mean of duplicates; +/- SD.

  • Colorimetric and Fluorometric examples of ADP Standard curve.

Protocols

References

This product has been referenced in:

  • Mitchell R  et al. A Targeted Mutation Disrupting Mitochondrial Complex IV Function in Primary Afferent Neurons Leads to Pain Hypersensitivity Through P2Y1 Receptor Activation. Mol Neurobiol N/A:N/A (2019). Read more (PubMed: 30689196) »
  • Müller WEG  et al. Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP. J Cell Sci 130:2747-2756 (2017). Read more (PubMed: 28687622) »
See all 6 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer

Thank you for your inquiry.

I would recommend the spin column filters (ab93349) over the PCA treatment for deproteinization. Theoretically the PCA treatment should be fine, but since we have not tested the outcome of this assay with such treated samples, I would go with the spin filter to be sure of no adverse effects.
I hope this information helps. Please contact us with any other questions.

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Answer

Thank you for contacting us.

Although the laboratory has not tested in the conditions you describe, using a reaction with an enzyme, they believe their is no compatibility issue and that it should work with some optimization. Same goes for the issue with CDP and IDP.

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
Answer

Merci pour votre appel de vendredi.

Le laboratoire m'a pu confirmer que vous pouvez procéder sans lescolonnes:

Pour le kit ADP ab83359, les échantillons lysés dans le"ADP assay buffer" peuventêtre aliquotes et congelées à-80°C avant utilisation. Ces échantillons ne doivent pas être filtrés.

Pour le kit ATP ab83355 les échantillons lysés dans le "ATP assay buffer" doiventêtre congelés rapidement en utilisant de l'azote liquide ou de la glacesèche si vous devez conserver les échantillons.C'est recommandé pour des meilleurs résultats, dedéproteinerou filtrer les échantillons. Comme vous n'avez pas de filtres, le laboratoire m'a confirmé que vous pouvez les déproteiner par précipitation:

Deproteinizing Sample Preparation Protocol:
The following protocol can be proportionally scaled up for preparation of larger or smaller sample volumes.

Protein Precipitation:
For biological samples with protein concentration less than ˜20 mg/ml (tissue homogenate, cell lysate, urine, etc.) take 550 µl of sample, mix with 100 µl of ice cold Perchloric acid in 1.5 ml microfuge tubes, vortex briefly to mix well, place on ice for 5 minutes. Centrifuge at 13,000g for 2 minutes. Accurately transfer 480µl of the supernatant to a fresh tube.

For serum and other very high protein concentration samples, take 400 µl of sample and mix with 100 µl of ice cold Perchloric acid, place on ice for 5 minutes. Centrifuge at 13,000g for 2 minutes. Accurately transfer 380 µl of the supernatant to a fresh tube.

Depending on the nature of the analyte, the samples in Perchloric acid may be frozen at -70°C for up to a month for storage at this stage.
Sample Neutralization:
Add 20 µl of ice cold 6 N KOH and mix to neutralize the sample and precipitate excess Perchloric acid. There may be some gas (CO2) evolution so vent the sample tube. Place on ice for 5 minutes. Spin briefly (˜1-2 min). Samples are now deproteinized, neutralized, and Perchloric acid has been removed. The samples may now be used in a variety of assays directly.




Note 1: The deproteinized samples have been diluted to 80% of the original concentration (quantitation results should be divided by 0.8 to correct measured values back to original sample concentrations). For serum samples, the dilution is to 76% so divide assay values by 0.76 to correct values to original sample concentrations.

Note 2: For further analysis of samples, if assay buffer is 0.1M or stronger, samples up to 50 µl may be used directly in 100 µl assay reactions. If lower concentration buffers are used in the assay, correspondingly smaller sample volumes should be used to maintain assay reaction pH without significant changes.

J'espère que ces informations vous sont utiles. Veuillez ne pas hésiter à nous recontacter si vous avez des autres questions.

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Answer

Unfortunately we do not give out our buffer components or compositions. Sorry about that.

Yes, it is very likely that the ATP in your buffer is giving the background. I would suggest doing a background control for each of the experimental samples, in order to account for this background in each of the readings.
Hope this information has been helpful for you. Please contact me again if you have any further questions.

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Answer

Thank you for contacting us.

I apologize for the wait, but I have not yet heard back from the lab. I have sent another email to follow up with them.

Thank you for your patience and understanding.

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Answer

Thank you for contacting us.

I have checked with the lab and their advice is as follows:

For most efficient results, we always recommend that the samples be made in the assay buffer provided with the kit. In case of that not being possible due to sample limitations, one can dilute it in the assay buffer as a last resort. However, this might compromise the results to a certain extent.

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
Answer

Thank you for contacting us.

I can confirm that the assay buffers in both the kits are different. These cannot be used interchangeably. If you are running out of buffers then let us know we can provide them separately.

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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https://www.abcam.com/abreviews

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Answer

Thank you for your questions. My colleague is out of the office today but I would like to forward the response that we received from the lab:

Unfortunately, these three kits have different Assay buffers and converting enzyme mixes, so they can not be shared.

I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

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Question
Answer

Thank you for your inquiry. This kit has not been tested with bacterial cells by the lab, however theoretically as long as the cells are prepared as indicated in the attached data sheet, it should work with such samples.  I hope this information helps. Please contact us with any other questions.

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