Overview

  • Product name

    Phosphate Assay Kit (Colorimetric)
    See all Phosphate kits
  • Detection method

    Colorimetric
  • Sample type

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

    Enzyme activity
  • Sensitivity

    > 1 µM
  • Range

    0.001 mM - 1 mM
  • Assay time

    2h 00m
  • Product overview

    Phosphate Assay Kit (Colorimetric) (ab65622) provides an easy, quick and simple method for measuring phosphate levels.


    The phosphate assay protocol uses a proprietary formulation of malachite green and ammonium molybdate which forms a chromogenic complex with phosphate ion giving an intense absorption band around OD = 650nm.


    Phosphate assay protocol summary:
    - add reaction mix to sample and standard wells
    - incubate for 30 min
    - analyze with a microplate reader


     

  • Notes

    This assay can be used with biological fluids but also inorganic samples such as algal blooms and water from run-off areas of high fertilizer use.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at room temperature. Please refer to protocols.
  • Components 500 tests
    Phosphate Reagent 1 x 15ml
    Phosphate Standard (10 mM) 1 x 500µl
  • Research areas

  • Relevance

    Phosphate is one of the most important of the inorganic ions in biological systems. It functions in a variety of roles. One of the most important roles is as a molecular switch, turning enzyme activity on and off through the mediation of the various protein kinases and phosphatases in biological systems. Phosphate is also of great importance in mineralization processes and is a primary stimulus of algal blooms frequently found in bodies of fresh water, due to run-off from areas of high fertilizer use.

Images

  • Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay (ab65622) and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem.
  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • Phosphate measured in tissue lysates showing quantity (µmol) per mg of extracted protein.

    Protein concentration for samples varied from 9 mg/mL to 14 mg/mL. Samples were diluted 400-4000 fold.

  • Phosphate measured in cell culture lysates showing quantity (µmol) per mg of extracted protein.

    Samples with the concentration of 1.3e7 cells/mL were used. Samples were diluted 400-4000 fold.

  • Phosphate measured in biological fluids showing quantity (µmol) per mL of tested sample. Samples were diluted 100-1000 fold.

Protocols

References

This product has been referenced in:

  • Zhou X  et al. Why SNP rs227584 is associated with human BMD and fracture risk? A molecular and cellular study in bone cells. J Cell Mol Med 23:898-907 (2019). Read more (PubMed: 30370607) »
  • Cliffe R  et al. Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme. Cell Rep 26:2140-2149.e3 (2019). Read more (PubMed: 30784595) »
See all 27 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Answer


I can confirm this kit has not been tested on samples containing polyphosphate. As long as there is phosphate ions in solution, this kit should detect the phosphate ions.

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Answer



I am very glad to hear that sonification of cells in Tris buffer gave you good results and less background than with a standard RIPA buffer.

I have made a note of this information for our lab as this is extremely useful information.

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Answer

With this kit ab65622, you can use any generic lysis buffer that you wish for preparing your sample. For example a RIPA buffer or NP-40 buffer is commonly used.

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Answer

Thank you for contacting us.



ab65622 is a colorimetric assay with a working range of 1-10uM concentration of phosphate. ab65622 provides enough materials to carry out 500 assays.

ab102508 is a more sensitive fluorometric assay with a working range of 0.4-10uM. ab102508 provides materials to perform 100 assays.



Glucose control: Since the fluorometric kit is 10-fold more sensitive, if there is glucose in the samples in comparable amounts with the phosphate (no specific amount can be suggested, depends upon relative amounts of glucose and phosphate in the sample), a glucose control is recommended for the purpose of proper background subtraction. For the colorimetric kit this should not be a problem.



A digestion step has not been tested in our protocol. If the customer wishes to test it, they can and there is a possibility that the total and the inorganic phosphate can be quantified. I suspect significant optimization of multiple parameters will be required to do so.



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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Answer

Thank you for contacting us.

The phosphate reagent will only interact with ionic phosphate in your samples, not the ADP and CDP.

Please let me know if you have any further questions.

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Question
Answer

Hello! I checked with the lab and they confirmed that we have never tried this kit with such samples, however theoretically it will be possible to use them.

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Answer

Thank you for contacting us.

The kit assays only for the free phosphate. Please make the standards as recommended in water. This is what you are diluting your samples with also.

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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Answer

Thanks for your query.
I got a reply from our lab regarding your questions:
"The short answer to your question is that you can use mouse plasma and frozen tissues with this assay kit. But there are a few considerations to keep in mind.

Although we have not tried EDTA derived plasma with this kit and have not heard any client complaints from such use, I would recommend your client to use Heparin instead of EDTA for plasma prep for this assy kit.

Fresh tissue is always preferable over frozen tissues. The success of using frozen tissue depends on how well the tissue was frozen and how long it has been in that state."

Hope this information has been useful for you. Please let me know if you have any other questions.

Thanks and have a very nice day.

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Answer

Thanks for the query.

Yes, this kit can be used for your purpose. Use any generic lysis buffer for making the extract. Use a Dounce homogenizer / sonicator for homogenization. Cell and tissue extracts/lysates also contain phosphates from the breakdown of nucleic acids, lipids, etc. Extraneous free phosphate should be removed from samples to be analyzed. Two commonly used methods of removing phosphate from samples are desalting columns and immunoprecipitation capture of the
Phosphate-generating enzymes being studied.
For the protocol, pleasesee the linkon our datasheet to the Protocol Booklet.
Hope this information has been helpful for you.

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Answer

Thank you for your reply.

I apologize, however there was a misunderstanding. I did not know that you were asking for the malachite green based assay used for assessing the activity of ab78432.

We do not knowwith which kitthe recombinant GRP78ab78432 activity has been tested. We have now asked the laboratory to receive these details and will update you on this.

The ab65622 is the only malachite green based kit we have in our catalog currently - this is why I cited this product.

Once again, please apologize for the confusion. We will write you again, as soon as we have more information. Thank you for your patience.

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1-10 of 13 Abreviews or Q&A

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