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Phosphate Assay Kit (Colorimetric) (ab65622)

Submit a review Q&A (13)References (42)
Functional Studies - Phosphate Assay Kit (Colorimetric) (ab65622)
  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell culture media, Cell Lysate, Other biological fluids, Plasma, Serum, Tissue Extracts
  • Sensitivity: 1 µM

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 product is manufactured by BioVision, an Abcam company and was previously called K410 Phosphate Colorimetric Assay Kit. K410-500 is the same size as the 500 test size of ab65622.

    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

  • Functional Studies - Phosphate Assay Kit (Colorimetric) (ab65622)
    Functional Studies - Phosphate Assay Kit (Colorimetric) (ab65622)Larissa S.Moniz, et al., Scientific Reports volume 7, Fig 5, Article number: 39985 (2017)
    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.
  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
    Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)

    Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
    Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)

    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.

  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
    Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)

    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.

  • Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)
    Functional Studies - Phosphate Colorimetric Assay Kit (ab65622)

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

References (42)

Publishing research using ab65622? Please let us know so that we can cite the reference in this datasheet.

ab65622 has been referenced in 42 publications.

  • Paparokidou C  et al. Phosphate availability and ectomycorrhizal symbiosis with Pinus sylvestris have independent effects on the Paxillus involutus transcriptome. Mycorrhiza 31:69-83 (2021). PubMed: 33200348
  • Matoo OB  et al. Temperature but not ocean acidification affects energy metabolism and enzyme activities in the blue mussel, Mytilus edulis. Ecol Evol 11:3366-3379 (2021). PubMed: 33841790
  • Regmi G & Dhakal IP Systemic levels of iron, phosphorus, and total protein in normocyclic versus repeat breeder Holstein Friesian crossbred cows of Kesharbag, Chitwan, Nepal. Vet World 13:2353-2357 (2020). PubMed: 33363326
  • Ye Y  et al. N-Terminal Ubiquitination of Amyloidogenic Proteins Triggers Removal of Their Oligomers by the Proteasome Holoenzyme. J Mol Biol 432:585-596 (2020). PubMed: 31518613
  • Bhat OM  et al. Medial calcification in the arterial wall of smooth muscle cell-specific Smpd1 transgenic mice: A ceramide-mediated vasculopathy. J Cell Mol Med 24:539-553 (2020). PubMed: 31743567
View all Publications for this product

Customer reviews and Q&As

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1-10 of 13 Abreviews or Q&A

Question

will this kit work with groundwater or rain samples?

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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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Question

I was just wondering if this kit detects polyphosphate? If not would it be suitable too hydrolyses polyphosphate using acid to form orthophosphate which then can be detected using this kit.

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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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Question

RIPA buffer made us a high background in the Analysis of phosphate assay (with cell lysates).

We modified the protocols and we found out if we do not use any lysis buffer, just kryolysis or sonification of the cells in the Tris buffer, is the best way how to prepare samples for this Analysis.

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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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Question

We ordered ab65622 phosphate assay kit. We would like to mesure aktivity of sulfotransferase in the cells.

In the sample preparation is to specified the lysis buffer for the cells.

Would you recommend me some for this Analysis?

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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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Question

Hello. I am writing about your phosphate assay kits (ab65622 and ab102508). We are mainly interested in determining the inorganic phosphates in red blood cell and platelet products from blood banks. I am wondering if you would recommend one kit over the other for this purpose, and if there might be any special considerations for these types of samples.



In the protocol for ab102508, it mentions that if a “significant amount of glucose” is present in the samples, then a glucose correction should be done. What would you consider to be a significant amount of glucose? In our red blood cell samples, for example, there might be up to 30 mmol/L glucose. Would this be enough to warrant a correction? Since this is not mentioned in the absorbance kit (ab65622), I presume that there is no interference in that procedure.



Finally, out of curiosity, would adding a digestion step allow us to be able to determine total phosphates, and then determine the organics by taking the difference between total and inorganic phosphate?

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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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Question



Inquiry: Does adenosine 5'-diphosphate, cytidine 5'-diphosphate or inosine 5'-diphosphate react with the phosphate reagent of Kit AB65622? I want to detect only inorganic phosphate as a product from a reaction that uses these nucleotides as substrates.

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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


Inquiry: Hello, I am a postdoc at U. Southern Calif. I use this phosphate assay kit to detect phosphate from hydrolysis of ATP by Na/K ATPase. I found that when I prepare the standard with just diH2O, it worked well. However, since my samples contain some ATP so I made my standard using the same buffer as my samples. My standard then became a flat line (all conc. had the same color). Does the phosphate reagent react both with free phosphate and ATP-bound phosphate? Thanks

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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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Question

I was wondering this this Phosphate Assay Kit (Colorimetric) (ab65622) can be used on mouse plasma (containing EDTA) or mouse tissue (frozen)?

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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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Question

I am a post-doc in Harvard Medical School. My porpose is to check the amount of phosphate uptake in the cells before and after certain treatment. Can I use this kit to for this purpose? What protocol do you recommend? Any special treatment for lysing the cell? Thanks.

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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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Question

With that said, any further details of the assay conditions used to test the recombinant GRP78 activity would be very helpful. I am planning to purchase this item and probably the kit as well for the purpose of conducting activity assays, and your working protocol seems like a great place to start for my own experiments.

Thanks,

That actually helps a lot, to know that this particular assay kit (ab65622) was used to assess the ATPase activity of the recombinant GRP78 product (ab78432). Perhaps that information could be added to the product information for the GRP78?

thanks,

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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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