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I have a doubt regarding the sample preparation for this kit.
According with the protocol, samples should be homogenized with Ice-
cold PBS and after that frozen with liquid nitrogen. However, I´m
going to measure a-KG levels in cultured cells -not in tissue-. Should
I make also the step with the liquid nitrogen? Could I add directly
the perchloric acid to the cells and after that make the
neutralization with the KHCO3?
Asked on May 29 2013
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.
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.
Answered on May 29 2013