Overview

  • Product name

    Alpha Ketoglutarate (alpha KG) Assay Kit
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

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

    Enzyme activity (quantitative)
  • Sensitivity

    = 0.01 nmol/well
  • Range

    0.01 nmol/well - 10 nmol/well
  • Assay time

    0h 30m
  • Product overview

    Alpha Ketoglutarate (alpha KG) Assay Kit provides a simple, sensitive and rapid means for quantifying alpha KG.


    In the alpha KG assay protocol, alpha-ketoglutarate is transaminated with the generation of pyruvate, which is used to convert a nearly colorless probe to a colored (λmax = 570 nm) and fluorescent (Ex/Em = 535/587 nm) product.


    The alpha-Ketoglutarate assay kit can be used to measure alpha-ketoglutarate in the range of 0.01 to 10 nmoles.


    Alpha Ketoglutarate assay protocol summary:
    - add reaction mix to standard and sample wells
    - incubate for 30 min
    - analyze with a microplate reader


     

  • Platform

    Microplate reader

Properties

Images

  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • Standard curve: mean of duplicates (+/- SD) with background reads subtracted

  • Alpha Ketoglutarate measured in biological fluids showing quantity (nmol) per mL of tested sample. Samples were diluted 2-18 fold 

Protocols

References

This product has been referenced in:

  • Wang J  et al. miR-9-5p inhibits pancreatic cancer cell proliferation, invasion and glutamine metabolism by targeting GOT1. Biochem Biophys Res Commun 509:241-248 (2019). Read more (PubMed: 30591220) »
  • Hong R  et al. Preventing BRCA1/ZBRK1 repressor complex binding to the GOT2 promoter results in accelerated aspartate biosynthesis and promotion of cell proliferation. Mol Oncol 13:959-977 (2019). Read more (PubMed: 30714292) »
See all 11 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Vielen Dank für Ihren Anruf.
Das Labor hat auf unsere Anfrage hin bestätigt, dass es wichtig ist, die Zellen mechanisch aufzuschließen, d.h. zu homogenisieren. Wie Sie richtig sagten, reicht PBS dafür nicht aus, sondern es empfiehlt sich eine Homogenisierung mit einem Dounce Homogenizer.
Vielen Dank, dass Sie uns auf diese Unklarheit hingewiesen haben. Wir werden das Protokoll in Kürze aktualisieren.
Ich hoffe, diese Informationen helfen Ihnen weiter, und ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Answer

The most efficient results will be had if the homogenate is made with the assay buffer provided with the kit. Rapidly homogenize ~2 x 10^6 cells with 100 µl of ice cold α-KG Assay Buffer using a dounce homogenizer. Deproteinate the samples so you can get rid of any interfering proteins including the enzymes. This is a small molecule analyte assay and cellular enzymes can interfere with the signal development

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Answer



There are two options for the sample preparation.

Either just rapidly homogenize cells with 100 μl of ice cold PBS or other buffer (pH 6.5-8) and then add 1-50 μl samples into duplicate wells of a 96-well plate and bring volume to 50 μl with Assay Buffer.

Or:

To prevent enzymes interfering with the assay use the perchloric acid/KOH protocol (step a to e). For cells, I can recommend to pellet them and then freeze them in liquid nitrogen.

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Answer

Thank you for contacting us. Did you compare the signal you obtained from the oxaloacetate with the results from a negative control? The control we recommend is testing without addition of the converting enzyme, in order to account for signal contributed by pyruvate and other probe reactants, and subtracting this value from the values obtained from the samples that are incubated with the converting enzyme.

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Answer

Thank you for your inquiry.

Yes, buffers with pH range 6.5 – 8 can be used. Just make sure that the RIPA buffer is within the recommendedpH.
I hope this information helps. Please contact us with any other questions.

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Answer

Thank you for contacting us.

It is possible to use the smaller sized spin column. The one thing to consider, though, is that the smaller the size of the filter, the harder it is to spin the column dry so that all the liquid is recovered. As a result, you may lose metabolite. We would recommend using the 10kD spin column (ab93349) for this reason.

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

This is a fluorescent assay and you will have to measure at two wavelengths: 535 nm and 587 nm. If you only have the 595 nm and not the 535 nm you will not be able to use the fluorescent measurements. However, this assay can also be measured colorimetrically (lmax = 570nm). So if your 595 nm filter has a bandwidth option of +/- 25, you can measure at the required 570 nm and be able to capture 100% of the signal.

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Answer

This kit can be used to measure alpha ketoglutarate in blood, plasma and Islets of Langerhans including tissue culture supernatants, urine, and serum samples.

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