Overview

  • Product name
    Pyruvate Kinase Assay Kit
  • Detection method
    Colorimetric/Fluorometric
  • Sample type
    Cell culture supernatant, Urine, Serum, Plasma, Other biological fluids, Tissue
  • Assay type
    Enzyme activity (quantitative)
  • Sensitivity
    = 0.1 mU/ml
  • Assay time
    0h 40m
  • Product overview

    Pyruvate Kinase Assay Kit ab83432 provides a simple, direct and automation-ready assay for measuring pyruvate kinase activity in cell culture supernatant, plasma, serum, tissue, urine and other biological fluids (not validated for use with whole blood).


    In the pyruvate kinase assay protocol, PEP and ADP are catalyzed by pyruvate kinase to generate pyruvate and ATP. The generated pyruvate is oxidized by pyruvate oxidase to produce color (absorbance 570 nm) and fluorescence (Ex/Em 535/587 nm). Since the increase in color or fluorescence intensity is proportional to the increase in pyruvate amount, the PK activity can be accurately measured.


    The kit detects 0.1 mU/ml pyruvate kinase.


    Pyruvate kinase assay protocol summary:
    - add samples and standards to wells
    - add reaction mix
    - analyze with microplate reader immediately and after 10-20 min

  • Notes

    Pyruvate kinase (PK, EC 2.7.1.40) is an enzyme involved in glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. Lack of pyruvate kinase will slow down the process of glycolysis which causes the disease known as pyruvate kinase deficiency.

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    OxiRed Probe Red 1 x 200µl
    PK Assay Buffer WM 1 x 25ml
    PK Enzyme Mix (Lyophilised) Green 1 vial
    PK Positive Control (Lyophilised) Blue 1 vial
    PK Substrate Mix (Lyophilised) Purple 1 vial
    Pyruvate Standard (100 nmol/µl) Yellow 1 x 100µl
  • Research areas
  • Relevance
    Pyruvate kinase (PK, EC 2.7.1.40) is an enzyme involved in glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. Lack of pyruvate kinase will slow down the process of glycolysis which causes the disease known as pyruvate kinase deficiency.
  • Cellular localization
    Cytoplasmic and Nuclear
  • Alternative names
    • CTHBP
    • EC 2.7.1.40
    • MGC3932
    • OIP3
    • OPA interacting protein 3
    • p58
    • PK
    • PK muscle type
    • PK1
    • PK2
    • PK3
    • PKL
    • PKLR
    • PKM
    • PKM2
    • PKR
    • PKRL
    • Pyruvate kinase 1
    • Pyruvate kinase 2/3
    • Pyruvate kinase isozyme R/L
    • Pyruvate kinase isozymes M1/M2
    • Pyruvate kinase liver and blood cell
    • Pyruvate kinase liver and RBC
    • Pyruvate kinase muscle
    • Pyruvate kinase type L
    • R type/L type pyruvate kinase
    • Red cell/liver pyruvate kinase
    • RPK
    • TCB
    • THBP1
    • Thyroid hormone binding protein cytosolic
    • Tumor M2 PK
    see all

Images

  • PC-3 and MCF-7 cells were treated with OA at the indicated doses and PK activity was determined 12 hr after OA treatment using ab83432. The average values of three independent experiments were shown as Mean ± SD. *, P<0.05, **, P<0.01.

  • Pyruvate measured in biological fluid after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per ml of tested sample.

  • Pyruvate measured in mouse tissue lysates after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per mg of tested sample.
  • Pyruvate measured in cell lysates after 2 min, 10 min and 20 min incubation time showing quantity (nmol) per 1 mln of tested cells.
  • Colorimetric standard curve: mean of duplicates (+/- SD) with background reads subtracted.
  • Fluorometric standard curve: mean of duplicates (+/- SD) with background reads subtracted.

  • Pyruvate kinase samples time line using ab83432.
  • Pyruvate standard curve using ab83432.

Protocols

References

This product has been referenced in:
  • Yoon YJ  et al. 2'-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2. Cancer Lett 434:42-55 (2018). Read more (PubMed: 30009856) »
  • Tanner LB  et al. Four Key Steps Control Glycolytic Flux in Mammalian Cells. Cell Syst 7:49-62.e8 (2018). Read more (PubMed: 29960885) »
See all 5 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer

Thank you for contacting us.

While we have not specifically tested ab83432 with PKM2, as long as the PKM2 isoform also catalyzes PEP and ADP to generate pyruvate and ATP, it will be detected by this kit.

The probe used in this kit is optimized for use with Ex/Em = 535/587. You should still be able to obtain a signal at Ex/Em = 550/590, but the signal to noise ratio may not be as good, which would lead to the kit being slightly less sensitive. I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us.

I would not recommend to use a normal cuvette for a spectrometer/Colorimeter because the volume you will use is much much smaller (50 ul to 1 ml or more). Or you will have to use much more to product which may be a problem when you want to use 200 samples.


However, if you could use cuvettes with a maximum of 100ul volume, and your Colorimeter is able to read the results from this micro-cuvettes, it may be possible, but only a little bit more expensive.


I hope this information is still 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 pyruvate kinase gene is mammalian.
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.

Unfortunately, the composition and their concentrations of each of these kit components is proprietary information, which I cannot divulge.

I am sorry for not being able to provide you any useful information in this case. Please do not hesitate to contact us if you need any more advice or information in other aspect.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your email.

Safety datasheets (SDS) are always available from the product page on our website.

The assay buffer contains Igepal, which may cause eye damage if it comes in contact with your eyes. That is the only hazardous material in the assay buffer.

I hope this helps. Please do not hesitate to contact us if you need anything further.

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Answer

Thank you for your patience. After reviewing your data, my colleague in the lab believes that the high background is likely caused by contamination in one or more of the reagents. Please let me know if the replacement kit is working better for you. If so, I will be happy to send you a second kit to compensate for the second unit that you ordered. If the new kit is also not working I will be happy to refund your original purchase.

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Answer

Thank you for your enquiry.

Theoretically the Pyruvate Kinase (PK) Assay Kit should work with isolated red blood cells (RBC). However, we have not purified RBC and tested it with this kit, therefore, we do not have any specific protocol recommendations. If the customer has a reliable way to isolate RBC, he can use these cells. Please be aware though that fresh whole blood coagulates which might negatively influence the isolation of RBC, and that through centrifugation not only RBC are pelleted, but also white blood cells are included.

Hence the isolation of RBC as well as the protocol for the PK Assay kit will have to be optimised by the customer in this case.

We aim to provide as much information as possible to customers, so I am sorry that this has not been possible on this occasion. I hope this information is nevertheless helpful to you. Should you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for contacting us.

The lab recommends incubating the standards for 20-30 minutes and using these readings to plot the standard curve. The sample readings for T1 and T2 should be such that they fall within the linear range of the standard curve.

Regarding the assay buffers, unfortunately the Pyruvate Kinase and Isocitrate Dehydrogenase assay buffers are not interchangeable.

I hope this helps, please let me know if you need any additional information.

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Question
Answer

Thank you for contacting abcam regarding assay buffers.

I have had a chance to speak with the lab about this. The assay buffers of both these kits are quite different and cannot be used interchangeably.

Please let me know if you have any questions.

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