Overview

  • Product name

    Ammonia Assay Kit
    See all Ammonia kits
  • Detection method

    Colorimetric
  • Sample type

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

    Quantitative
  • Sensitivity

    20 µM
  • Assay time

    1h 00m
  • Product overview

    Ammonia Assay Kit (ab83360) provides a rapid, simple, sensitive, and reliable assay for ammonia and ammonium.


    In the ammonia assay protocol, ammonia and ammonium are converted to a product that reacts with a probe to generate color (λmax = 570 nm) which can be easily quantified by plate reader. The kit can detect 1 nmol (~20 µM) of total ammonia and ammonium, which is much more sensitive than measuring ammonia with a NADPH based assay.


    Ammonia assay protocol summary:
    - add samples and standards to wells
    - add reaction mix and incubate for 60 min at 37ºC
    - analyze with microplate reader

  • Notes

    Ammonia assays measure the total level of the ammonium ion and of ammonia in a sample.

    At a physiological pH, nearly all ammonia exists as ammonium in solution.

    The chemical equation that drives the relationship between ammonia and ammonium is: NH3 + H2O ↔ NH4+ + OH-

    When the pH is low, the reaction is driven to the right, and when the pH is high, the reaction is driven to the left. In general, at around room temperature, at a pH less than 6.0, the proportion of ammonium-N plus ammonia-N as NH3 is very low and as NH4+ is very high. At a pH around 8.0 (the ammonia assay buffer pH), the proportion as NH3 is 10 percent or less.

    This is our most popular ammonia assay kit, ammonia assay kit ab102509 uses an alternative non-enzymatic method.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    Ammonia Assay Buffer WM 1 x 25ml
    Converting Enzyme (Lyophilized) Blue 1 vial
    Developer Orange 1 vial
    Enzyme Mix (Lyophilized) Green 1 vial
    NH4Cl Standard Yellow 1 x 100µl
    OxiRed Probe in DMSO Red 1 x 200µl
  • Research areas

  • Relevance

    Ammonia is an important source of nitrogen for living systems. Nitrogen is required for the synthesis of amino acids, which are the building blocks of protein. Ammonia is a metabolic product which is created through amino acid deamination. It plays an important role in both normal and abnormal animal physiology like normal animal acid/base balance.

Images

  • Zhiyuan Li et al. used ammonia assay kit ab83360 to examine the build-up of ammonia in old cell culture medium containing glutamine and how this might induce autophagy in cultured cells. 

    The ammonia assay was used to measure the ammonia concentration in cell culture medium containing Glutamine (medium alone, without cells) which was stored at 4°C or at 37°C for between 1-7 days. They found that ammonia levels increased significantly in the medium stored at 37°C.

  • Kyle Wilson et al. used ammonia assay kit ab83360 to examine ammonia levels in liver tissue in response to P. chabaudi infection. They found that in IL-10 KO mice, ammonia levels were not elevated above WT during P. chabaudi infection.

    WT and IL-10 KO mice were infected with P. chabaudi and monitored during the peak of infection. WT mice were sacrificed at the peak of infection (day 10 p.i.) and IL-10 KO mice upon severe morbidity as determined via SHIRPA score.

  • Ammonia assay performed with cell culture medium (range of dilution 1:10-1:200) and control medium (range of dilution 1:1-1:20), background signal subtracted (duplicates +/- SD).

  • Ammonia assay performed with mouse tissue lysates (mg of extracted protein), background signal subtracted (duplicates +/- SD).

  • Ammonia assay performed in cell lysates, background signal subtracted (duplicates +/- SD).

  • Ammonia assay performed in biological fluids (range of dilution 1:1-1:100), background signal subtracted (duplicates +/- SD).

  • Example of ammonia assay standard curve using ab83360

Protocols

References

This product has been referenced in:

  • Xu S  et al. An Integrative Systems Biology and Experimental Approach Identifies Convergence of Epithelial Plasticity, Metabolism, and Autophagy to Promote Chemoresistance. J Clin Med 8:N/A (2019). Read more (PubMed: 30736412) »
  • Khoja S  et al. A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia. J Inherit Metab Dis N/A:N/A (2019). Read more (PubMed: 30835861) »
See all 15 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Answer

The kit detects Ammonia which would exist as ammonium ion in solution. The detection principle is based on reaction with ammonium ion in solution. This ammonium ion population includes free pre-existing cellular ammonium and the ionized form of ammonia. To subtract background from pre-existing ammonium, run a parallel reaction excluding the converting enzyme. This would also allow subtraction of any interference from endogenous Pyruvate.

This kit therefore measures total ammonium in solution. At physiological pH, nearly all ammonia will exist as ammonium in solution.

The chemical equation that drives the relationship between ammonia and ammonium is:

NH3 + H2O ↔ NH4+ + OH-

When the pH is low, the reaction is driven to the right, and when the pH is high, the reaction is driven to the left. In general, at around room temperature, at a pH less than 6.0, the proportion of ammonium-N plus ammonia-N as NH3 is very-very low and as NH4+ is very-very high. At a pH around 8.0 (the assay buffer pH), the proportion as NH3 is 10 percent or less. Laboratory methods measure ammonium-N plus ammonia-N. It is very difficult to directly determine the activity of aqueous ammonia, so instead the surrogate of ammonium-N plus ammonia-N is used.

Controls:

1. If any buffer used in the experiment has ammonium salt (e.g. NH4Cl), the buffer must be used to assay how much ammonium is in it. This number can be subtracted from the cells + buffer number to know how much ammonium is in the cells.

2. Also, the zero standard gives the background reading for contamination from ammonia in air.

3. Omitting the converting enzyme gives background from pyruvate which is an intermediate in the colour development reaction.

Read More

Answer

Thank you for contacting us.
In response to your questions on this kit, the pH of the sample should be ˜7-8. To use mouse stool sample, add assay buffer to sample. Mix it completely either by pipetting up & down, sonicating or vortexing. Centrifuge to remove insoluble material & use the supernatant to do the assay according to the protocol mentioned in the datasheet.

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

This kit measures the total ammonia + ammonium concentration in your samples. Ammonium does not interfere with this assay.

The actual method and way the kit does this is proprietary information and so I am unable to provide more information.

Please let me know if there is anything else I can help you with.

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Question
Answer

Phenol red containing media can interfere with the results of this assay. Diluting the media samples with assay buffer may help reduce the concentration of the phenol red thus minimizing the background.

My advice is to switch to medium that is phenol red-free. I think you mentioned that you were using RPMI, which is available in that formulation.

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COMPATIBILITY OF THE KIT WITH ORGANIC SOLVENTS

Excellent Good 4/5 (Ease of Use)
Abreviews
The ammonia determination is accurate for samples of tissue extracted in methanol:acetonitrile:water (5:3:2) solution, provided that the samples are diluted 1:5 in assay buffer. The reaction efficiency decreases when the ratio between organic solvents and assay buffer increases. The reaction does not occur if the samples in organic solvents are not further diluted in assay buffer.
Figure Legend. The standard curve has been generated by mixing in each well 10 microliters of methanol, acetonitrile, water, solution containing known amounts of ammonia, to 40 microliters of assay buffer. The reaction started with the addition of 50microliters/well of reaction mix. Samples were incubated at 37 degrees for 1 hour.

Abcam user community

Verified customer

Submitted Jul 27 2016

Answer

For this kit, we prefer measuring from fresh samples for best results but cell culture media can be centrifuged to remove any debris and then stored at -80C for future assay.

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Abreviews
We had good results using the ABCAM ammonia kit for the detection of NH3 in our cell lysates. However, we wanted to see the distribution of NH3 in the intact cells to determine the intercellular variation. We were able to adapt the kit to allow for the in situ detection of ammonia. We chose two cell lines one strongly + for NH3 and one -. The key was to use formalin fixed cells AND to digest in protease prior to use. Under these conditions, we got an excellent signal in the + cells and no signal in the - cells.

Abcam user community

Verified customer

Submitted Dec 27 2013

Answer

For serum or plasma samples, there are no special requirements for preparation of serum or plasma samples for the ammonia assay.

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Question
Answer

Thank you for contacting us.

This kit has been tested on mouse brain, heart, liver kidney and muscle tissues. Please see figure 3 in the protocol booklet for more information.
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.

Yes, storage at -80 C until you are ready to perform the assay will be fine.

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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1-10 of 12 Abreviews or Q&A

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