Overview

  • Product name
    Hexokinase Activity Assay Kit (Colorimetric)
    See all Hexokinase kits
  • Detection method
    Colorimetric
  • Sample type
    Serum, Tissue, Adherent cells, Suspension cells, Tissue Extracts
  • Assay type
    Enzyme activity (quantitative)
  • Sensitivity
    < 0.1 mU/well
  • Assay time
    1h 00m
  • Species reactivity
    Reacts with: Mammals, Other species
  • Product overview

    Hexokinase Activity Assay Kit (ab136957) uses a simple, sensitive and rapid procedure which can detect hexokinase activity to below 0.1 mU/well.


    In the hexokinase assay protocol, glucose is converted to glucose-6-phosphate by hexokinase.  The glucose-6-phosphate is then oxidized by glucose-6-phosphate dehydrogenase to form NADH. The NADH reduces a colorless probe to a colored product with strong absorbance at 450 nm.


    Hexokinase activity assay protocol summary:
    - add samples and standards to wells
    - add reaction mix and incubate for 20-60 min
    - analyze repeatedly with a microplate reader during the incubation

  • Notes

    Hexokinases (HK) are found in many organisms including bacteria, plants and mammals and play an important role in glucose metabolism. Hexokinases phosphorylate glucose and generate glucose-6-phosphate for glycolysis. Hexokinases have four isoforms (HK-I, II, III and IV). HK-I, HK-II and HK-III have low Km, while HK-IV has 100 fold high Km.

  • Platform
    Microplate reader

Properties

Images

  • Hexokinase Activity measured in cell lysates showing quantity (nmol) per 1 mln cells after 35 min of incubation.

    Samples with the concentration of 5e6 cells/mL were used. Samples were diluted 1-27 fold.

  • Hexokinase Activity measured in tissue lysates showing quantity (nmol) per mg of extracted protein after 35 min of incubation.

    Protein concentration for samples varied from 13 mg/mL to 43 mg/mL. Samples were diluted 1-9 fold.

  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted
  • NADH standard curve (a). Hexokinase activity in Positive Control and Jurkat cell lysate (40 µg) (b).

Protocols

References

This product has been referenced in:
  • Yao J  et al. By blocking hexokinase-2 phosphorylation, limonin suppresses tumor glycolysis and induces cell apoptosis in hepatocellular carcinoma. Onco Targets Ther 11:3793-3803 (2018). Read more (PubMed: 30013360) »
  • Bramer SA  et al. Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy. Reprod Biol Endocrinol 15:4 (2017). Functional Studies ; Horse . Read more (PubMed: 28056994) »
See all 4 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer



When preparing these samples please make sure to keep these on ice throughout the preparation procedure. Fresh samples will give you the best results with this kit, however, frozen samples can also be used. If freezing your samples, make sure to snap-freeze them at -80ºC in order to preserve them before using them with this kit.

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Answer

We have not specifically tested the kit with plasma sample however, it should work with mammalian samples including plasma. It should not affect the sensitivity or accuracy of the test.

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Answer

This kit is based on function of the Hexokinase enzyme (conversion of glucose to glucose6phosphate is measured) so this kit does not distinguish between the isoforms.

HKI, HKII and HKIII have low Km, while HKIV has 100 fold high Km. Hence it is very likely that you will measure hexokinase activity from I, II and III and small contribution if any from HK IV depending on the relative amounts of these isoforms in your sample.

HK-II is the predominant form in adipose and muscle cells. HKII is insulin-sensitive. So if the samples are from adipose tissue or muscle the customer will measure HK-II activity. Our positive control is HK-II from Bacillus sp.

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Answer

I can confirm the the Hexokinase substrate kit component included in the kit is glucose. This component was recently re-labeled. The error in the naming will be fixed shortly and a new protocol will be available for download from the website shortly. Unfortunately, the concentration of these components is proprietary information.

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Question
Answer

Yes, this buffer contains NP-40.

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Question
Answer

The assay buffer contains NP-40.

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Answer

I apologize for any misunderstanding regarding the unit label for this kit. I am glad you read through the documentation. You are correct in your assessment of how many actual samples can be tested using this kit, after taking into account controls and blanks.

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Answer

Unfortunately, the concentration of these components is proprietary information.

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Answer

Thank you for contacting us.

I have spoken with the lab in regards to these issues. While the positive control information is proprietary, I can confirm that this is a protein control.

Regarding separate purchase of the kit's developer, I am sorry but this cannot be offered a this time. I do apologize for the inconvenience.

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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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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