Overview

  • Product name

    Citrate Assay Kit
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

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

    Quantitative
  • Sensitivity

    > 0.002 mM
  • Range

    0.002 mM - 10 mM
  • Assay time

    0h 40m
  • Product overview

    Citrate Assay Kit ab83396 provides a simple, sensitive and rapid means of quantifying citrate in biological samples.


    In the citrate assay protocol, citrate is converted to pyruvate via oxaloacetate. The pyruvate is quantified by converting a nearly colorless probe to an intensely colored (570 nm) and fluorescent (Ex/Em, 535/587 nm) product.


    The citrate assay kit can detect 0.1 to 10 nmoles (~2 µM-10 mM) of citrate.


    Citrate assay protocol summary:
    - add samples and standards to wells
    - add reaction mix and incubate for 30 min at room temp
    - analyze with microplate reader

  • Notes

    Citric acid (HOOC-CH2-C(-OH)(-COOH)-CH2-COOH) is a key intermediate in the TCA cycle which occurs in mitochondria. It is formed by the addition of oxaloacetate to the acetyl group of acetyl-CoA derived from the glycolytic pathway. Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis.

  • Platform

    Microplate reader

Properties

Associated products

Images

  • Fluorimetric standard curve: mean of duplicates (+/-SD) with background readings subtracted.

  • Citrate measured fluorimetrically in various biofluids showing concentration (micromolar).

  • Citrate standard curve generated using this kit protocol

Protocols

References

This product has been referenced in:

  • Hogan SE  et al. Mesenchymal stromal cell-derived exosomes improve mitochondrial health in pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 316:L723-L737 (2019). Read more (PubMed: 30652491) »
  • Ren JG  et al. Citrate Suppresses Tumor Growth in Multiple Models through Inhibition of Glycolysis, the Tricarboxylic Acid Cycle and the IGF-1R Pathway. Sci Rep 7:4537 (2017). Functional Studies . Read more (PubMed: 28674429) »
See all 4 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Answer

Urine is a liquid and therefore doesn't need to be homogenised. It is true that enzymes in samples may interfere with the assay which is why we suggest deproteinizing samples using a perchloric acid/KOH protocol or 10 kd molecular weight cut off spin columns. However, normal urine is relatively free of protein and can be analyzed directly without the need of deproteinisation.

The exception is if you a studying diseases wherein proteinuria may be expected, in these cases, deproteinization should be performed and parallel samples of control urine from normal subjects should be treated in the same manner.

Using our kit, you can measure the concentration of urine in your samples. The concentrations are derived using a linear regression equation obtained by fitting a trendline in Excel (ask Excel to show the equation and the R2 on the graph-but do not ask it to set the y-intercept at zero). The R2 value is a measure of how well the data can be fit by a straight line (good fits are ˜ 0.99 and less than 0.98 is considered unacceptable). The linear equation is y = mx + b; where y = absorbance or RFU, x = sample amount, m = slope and b = y-intercept (the value at x = 0). One plots the standards after correcting for the reagent blank (i.e., 0 standard). The sample values must also be corrected for the reagent blank. In some cases, a sample background correction is also required. In addition, the sample absorbances or RFU’s must fall within the linear range of the standard curve (that is why we recommend testing a few sample dilutions). The calculations are then straight forward. Using the linear regression equation as follows: (where the sample readings are the reagent blank corrected values (also in some cases background corrected)

Concentration = Ay / Sv (nmol/μl; or μmol/ml; or mM)

Where: Ay is the amount of citrate (nmol) in your sample from the standard curve.
Sv is the sample volume (μl) added to the sample well.

I assume you have already calculated 5.16 nmol/well from your standard curve using the linear equation?

You now need to multiply 5.16 nmol/well by the sample volume taking into account any pre-dilution before adding the sample to the assay well.

For example, if y = 0.5 x + 0.001, the standard curve was in nmol/well, the sample was pre-diluted 5-fold then 2 µl was used and gave an absorbance of 0.2 then one would get:

Sample concentration = [(0.2 – 0.001)/(0.5 x 2)] x 5 = 0.995 nmol/µl or 0.995 µmol/ml or 0.995 mM

From what I understand, you want to determine the amount of citrate in mg from this concentration, is that correct?

If so, then this should be done using standard conversion formulae and is not described in the protocol booklet as this is not directly related to the kit.

In this case you would have to find the no of moles in your sample using the following equation:

no.of moles = concentration x volume

From this you can then find the mass of citrate in your samples using the following equation:

no.of moles x molecular weight of citrate = mass

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Answer

The lab has confirmed that the probe and all the reconstituted lyophilized components can be stored in aliquots at-20C. If never frozen and thawed, it is possible that the components will still work beyond 2 months.


The Urine can be stored at -20C. Please be aware that before analysis they have to be brought to room temperature and then centrifuged at 1400 x g for 10 min to remove any particular matter. Usually, if never frozen and thawed, urine samples should last for a month. However, it is difficult to say exactly for how long they can be stored. I would not suggest storing to assay citrate for too long.



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Answer

This kit can detect 0.1 to 10 nmoles (˜2 µM-10 mM) of citrate in a variety of samples. It can detect sodium citrate. In fact that is used as the standard.

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Answer

Thank you for your enquiry.
The optimum dilution factor depends on the cell type, the concentration of citrate in the cells and the overall quality of the lysate. This will need to be individually determined.
I would recommend making a series of dilutions over a broad range (e.g. 1 - 50ul per well) to make sure you get the best results which would read within the linear range of the standard curve. It is best to make up the dilutions first in a micro-centrifuge tube and then use the same volume of diluted sample in the wells to avoid pipetting errors.
I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

Thank you for contacting us and your interest in our products.

I am sorry but I am not sure of the property you are seeking. I am not familiar with the "CoV". In order to help further in your query, could you please clarify this further?

I look forward to receiving your reply.

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Answer

Thanks for your reply and your suggestions. I'll be sure to pass them on to the lab.

Please let me know if you have any further questions.

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Answer

The orbital shaker is not requiredto run this assay.Sorry for the confusion. You could, however, use it when incubating reagents in the microtiter plate if you wish but this is not required.

We can likely make the assay buffer available separately. Please let me know if you are you are interested in purchasing the buffer as a separate reagent and I will look into having it added to the catalog.
You can use a Dounce homogenizer with the tissue. See this link for an example of a dounce homogenizer for use with this volume: http://www.thomassci.com/Equipment/Grinders/_/MICRO-TISSUE-GRINDER-KIT?q=Dounce%20Homogenizer.

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

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Answer

Thank you for your enquiry.

I am sorry to confirm this kit has not yet been tested with bone samples, so we would not be certain how it would work. I would suggest that the protocol can be followed from the deproteinizing stage.

Here are a few references which may be helpful:

- Jones, A. M. et. al. The phytopathogen Pseudomonas syringae pv tomato DC3000 has three high affinity iron scavenging systems functional under iron limitation but dispensable for pathogenesis. J. Bacteriol., Mar 2011; 10.1128/JB.00069-10

- Jones, A. M. et. al. The Phytopathogen Pseudomonas syringae pv. tomato DC3000 Has Three High-Affinity Iron-Scavenging Systems Functional under Iron Limitation Conditions but Dispensable for Pathogenesis. J. Bacteriol., Jun 2011; 193: 2767 - 2775.

We aim to provide as much information as possible to customers, and I am sorry there is no further data or information regarding bone samples to send to you on this occasion.

If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for contacting us.

The assay buffers in each of these kits is different. The buffers are optimized in order to get the best results. Please use the assay buffer for the sample, specific to each 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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Answer

Add 4 M perchloric acid to a final concentration of 1 M, vortex briefly and incubate on ice for at least 15 min. Centrifuge at 13,000xg for 2 min at 4oC and transfer supernatant to fresh tube. Excess perchloric acid is precipitated as KClO4 by adding an equal volume of ice-cold 2M KOH and vortexing briefly. Centrifuge at 13,000xg for 15 min at 4oC and collect supernatant. You may wish to measure protein concentration before and after deproteinization to confirm protein has been removed. I hope this is helpful. Please contact me again if you have any further questions.

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1-10 of 18 Abreviews or Q&A

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