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Pyruvate Assay Kit (ab65342)

Reviews (2)Q&A (23)References (77)
Functional Studies - Pyruvate Assay Kit (ab65342)
  • Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric
  • Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric
  • Functional Studies - Pyruvate Assay Kit (ab65342) -Colourimetric
  • Functional Studies - Pyruvate Assay Kit (ab65342)
  • Functional Studies - Pyruvate Assay Kit (ab65342)

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric/Fluorometric
  • Platform: Microplate reader
  • Assay time: 40 min
  • Sample type: Cell culture extracts, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine
  • Sensitivity: 1 µM

Overview

  • Product name

    Pyruvate Assay Kit

  • Detection method

    Colorimetric/Fluorometric

  • Sample type

    Urine, Serum, Plasma, Cell culture extracts, Other biological fluids, Tissue Extracts

  • Assay type

    Quantitative

  • Sensitivity

    > 1 µM

  • Range

    1 µM - 200 µM

  • Assay time

    0h 40m

  • Product overview

    Pyruvate Assay Kit (Colorimetric/Fluorometric) ab65342 provides a simple, direct and automation-ready procedure for measuring pyruvate concentration in various biological samples such as blood, cells, culture and fermentation media, etc.


    In the pyruvate assay protocol, pyruvate is oxidized by pyruvate oxidase via enzyme reactions to generate color (λ= 570 nm) and fluorescence (at Ex/Em = 535/587 nm). Since the color or fluorescence intensity is proportional to pyruvate content, the pyruvate concentration can be accurately measured.


    The kit detects 1-200 µM pyruvate.


    Pyruvate 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

    This product is manufactured by BioVision, an Abcam company and was previously called K609 Pyruvate Colorimetric/Fluorometric Assay Kit. K609-100 is the same size as the 100 test size of ab65342.

  • Platform

    Microplate reader

Properties

Images

  • Functional Studies - Pyruvate Assay Kit (ab65342)
    Functional Studies - Pyruvate Assay Kit (ab65342)Kaur, Gursharan et al., Scientific reports vol. 7,1 11645., Fig 2, doi:10.1038/s41598-017-12004-3
    Pyruvate levels under manganese stress and iron supplementation were measured with ab65342 and the results were plotted as a function of normalized absorbance at 450 nm per milligram of cellular proteins.
  • Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric
    Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric

    Pyruvate measured fluorometrically in biological fluids showing quantity (nmol) per mln of tested cells

  • Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric
    Functional Studies - Pyruvate Assay Kit (ab65342) - Fluorometric

    Pyruvate measured fluorometrically in biological fluids showing quantity (nmol) per mL of tested sample

  • Functional Studies - Pyruvate Assay Kit (ab65342) -Colourimetric
    Functional Studies - Pyruvate Assay Kit (ab65342) -Colourimetric

    Pyruvate measured colourimetrically in biological fluids showing quantity (nmol) per mL of tested sample

  • Functional Studies - Pyruvate Assay Kit (ab65342)
    Functional Studies - Pyruvate Assay Kit (ab65342)

    Pyruvate colorimetric standard curve.

  • Functional Studies - Pyruvate Assay Kit (ab65342)
    Functional Studies - Pyruvate Assay Kit (ab65342)

    Pyruvate fluorometric standard curve.

References (77)

Publishing research using ab65342? Please let us know so that we can cite the reference in this datasheet.

ab65342 has been referenced in 77 publications.

  • Paruchuru LB  et al. The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells. Cells 11:N/A (2022). PubMed: 35159398
  • Wen YC  et al. Pyruvate kinase L/R links metabolism dysfunction to neuroendocrine differentiation of prostate cancer by ZBTB10 deficiency. Cell Death Dis 13:252 (2022). PubMed: 35306527
  • Yi B  et al. Circular RNA PLCE1 promotes epithelial mesenchymal transformation, glycolysis in colorectal cancer and M2 polarization of tumor-associated macrophages. Bioengineered 13:6243-6256 (2022). PubMed: 35349390
  • Brashears CB  et al. Malic Enzyme 1 Absence in Synovial Sarcoma Shifts Antioxidant System Dependence and Increases Sensitivity to Ferroptosis Induction with ACXT-3102. Clin Cancer Res 28:3573-3589 (2022). PubMed: 35421237
  • Cacciola NA  et al. Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer. Int J Mol Sci 23:N/A (2022). PubMed: 35955595
View all Publications for this product

Customer reviews and Q&As

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1-10 of 25 Abreviews or Q&A

Pyruvate assay is very quick and easy to use

 Excellent Good 4/5 (Ease of Use)
Abreviews
abreview image
We used this pyruvate assay to detect pyruvate in cell pellets and the kit was very simple to use and very fast. Samples likely need to be diluted if deproteinized, and if the concentration range is unknown, an additional one or two standards above or below the recommended standards can be used to cover a greater dynamic range. Care should be taken to wash all excess media out if the media contains excess pyruvate as a media supplement.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jan 16 2020

easy to use

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
Assay was performed in cancer cell lines. Instructions were easy to follow with good results
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Oct 22 2018

Question

I want to use this kit to detect pyruvate created by an enzyme. In order to induce the pyruvate reaction I add magnesium to my enzyme and quench with EDTA. Following, I would use the kit to quantify the amount of pyruvate produced. I was wondering if there are any metallic ions or other kit components that would be incompatible with the magnesium or EDTA.

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Answer

The laboratory responded that EDTA can interfere with the enzymatic reaction in this assay unless it can be diluted out to bare minimal.

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Question

Customer kindly contacted us inquiring use of this product in yeast and e.coli samples.

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Answer

I have spoken with the lab regarding the use of this kit in yeast and bacteria. They agree that while we have optimized this assay protocol for samples of mammalian origin, theoretically this assay should work with samples from multiple origins including bacteria and yeast. Since we have not done it ourselves, I do not know if the assay protocol is the most optimized for such samples, so you may have to do some optimization to get the most efficient results.

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Question

please i want to know sample appropriate for the assay is it whole blood or serum or what? and do i have to do some preparations for the sample before the assay?

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Answer


You can use plasma samples with this assay. Optimal dilutions have to be determined using a pilot experiment with different volumes.

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Question

We are using human and mouse urine that has been filtered to remove protein (as suggested by the kit insert) and then frozen once at -70° before assaying. It appears there is some analyte in the urine that inhibits the assay. We have assayed plasma and kidney cortex without any issues (other than a reduction in pyruvate in frozen samples). If you look at the two attached pictures you will see the fluorescent scans that exhibit the urine inhibition we have observed.   On the first plate (Image 1: Pyruvate - different dilutions mouse urine) I first became aware of a potential problem when trying to optimize the assay for urine using several dilutions of three different urine sample types. The normal (and most concentrated) urine is where we see the most inhibition. I hypothesize that what ever is causing the inhibition is the most concentrated in that type of sample. As the normal urine sample becomes more dilute, the fluorescence increases. So, although it appears that the urine collected from mice with disease have higher pyruvate using the 1:4 dilution, it is the opposite at the 1:400 dilution.   I then decided to check the theory that something in the urine was inhibiting the assay (see second image: pyruvate assay urine cortex plasma). Normal human urine was collected and immediately ran for pyruvate (both filtered and unfiltered with no difference). The urine was diluted with the pyruvate buffer and standard from the kit was added. As you can see, the standard curve was completely inhibited in 1:4 diluted urine and again in 1:100 diluted urine (although not completely at this dilution).   Has your lab tested the kit specifically for pyruvate in urine? om: us.technical@abcam.com [us.technical@abcam.com]

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Answer

Uric acid in urine is going to strongly interfere with the assay so besides doing the normal standard curve, the lab recommends to also do standard curve in the presence of X volume of diluted urine (may be 4-15 ul depends). Due to the presence of uric acid in the urine, the slope of standard curve with the urine will be different. Subtract the 0 standard from 0 Standard with urine (∆OD = 0 Standard with urine – 0 Standard). Apply this ∆OD to slope of standard curve with the urine to get pyruvate conc. in urine sample

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Question

Customer kindly contacted us as the pyruvate assay buffer when thawed had flake precipitate out. Should this be of concern? What is the recommended way to resuspend these? Would heating to 37C adversely affect the buffer?

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Answer

The lab has advised me that you can keep the buffer at 37°C for some time to see if it helps. The 37°C for a short time should not adversely affect the assay; in fact many clients do keep the buffer bottle at this temp water bath while thawing it out. If you are unable to get these flakes back into solution please let us know.

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Question

A. General Information
- Catalogue number: ab83429; ab65342
- Lot number:
- Purchase order number/ order date or preferably the Abcam order number:
- The kits were stored at -20C.
B. Description of the problem
- Are the signals you obtained with control or standard satisfactory? (Yes);
There was no problem in
preparation of lactate and pyruvate standard curves, but the test
sample wells didn't produce colour as the color of standard though I
have used 50ul of samples without dilution. However, I cannot use
10kDa spin column for deproteinization.Instead, I have used 8%
perchloric acid correctly.
- The sample readings didn't produce colour totally and even lost the
reaction mix colour.
- The blank readings are expected and ok.
C.The problem I think is what I didn't use spin column and used 8%
perchloric acid for deproteinization which inhibits the enzymes
present in the kits.
D. Sample
- Sample Type is whole blood which is deproteinized with 8% perchloric acid
- Sample Species ( human)
- Storage (at -20C)
- Sample is treated by 8% perchloric acid immediately.
- Sometime proteins in samples interfere with readings. Have you
removed proteins?
- Sample preparation
Draw 2 ml of blood and add to sodium floride and potassium oxalate
tube and immediately pour to 2 ml of premeasured chilled 8% perchloric
acid. And centrifuge at 3000g for 10 min for 2 times and get the
supernatant and freeze at -20C.
E. Protocol used:
I Follow the protocol except using of spin column
G. Have you used the same kit successfully before?
I have never used this kit before.
H. Do you obtain the same results every time?

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Answer

Thank you for taking the time to complete our questionnaire and providing this further information. I am sorry to hear you have had difficulty obtaining satisfactory results from our D-Lactate Assay Kit (Colorimetric) (ab83429) and our Pyruvate Assay Kit (ab65342).

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I appreciate the time you have spent to do so.

As the standard curve is working well, in this case I would be concerned about the sample preparation. I can confirm I would expect the deproteinisation with PCA will degraded the proteins and the acidic environment will inactivate all added enzymes as already suggested. If it is necessary for this experiment to use PCA we would recommend to neutralise the PCA before proceeding (for example potassium hydroxide). Can you confirm if this has been done?

The 10kDa spin column separates out the analytes from any protein sample without degradation of proteins. For that reason we would highly recommend to use Spin columns for the sample preparation if possible at all.

I hope this information is helpful for you. Please do not hesitate to contact me if you have further problems or questions.

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Question

Thanks for your reply. I would also like to know if there is a guideline for the period time that the cells need to be lysed in the assay buffer for complete lysis to occur.

Regards,

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Answer

Thank you for contacting us.

Start with ˜2X106 cells, suspend the cell pellet 500 μl (or ˜4 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

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

What is the minimum amount of assay buffer that could be used to prepare lysates?

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Answer

Thank you for your email.

We recommend using this kit with 2x106 cells per 500ul of assay buffer. This cell number however is for guidelines only; the number should be optimized as it varies based on cell line type.

One way to prepare cell lysates is by adding 4 times the assay buffer to cell culture media as recommended on the datasheet. Alternatively cells can be centrifuged and then assay buffer could be added - please use the above given cell number and volume to scale down the assay buffer volume as per your requirement.

I hope this information will be helpful. Should you have any other question please do not hesitate to contact me.

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1-10 of 25 Abreviews or Q&A

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