Alpha Ketoglutarate (alpha KG) Assay Kit (ab83431)

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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.
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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

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.

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.

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.

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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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.

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