Hello,
I recently performed Kit based ELISA for 2,3 DPG on plasma samples of control and hypoxia treated rats. [CAT. NO. E1562Ra, Bioassay technology laboratory]
However, the results I obtained were uniform.
My main question is, is it technically right to perform ELISA on 2,3 DPG which is an isomer of 1,3 DPG, which itself is a intermediate in glycolysis.
If it is, how does the kit differentiate between 1,3 DPG and 2,3 DPG?
The selectivity of your antibody should be described in its documentation, if not, ask the manufacturer.
To specifically assay 2,3-BPG in the presence of 1,3-BPG I'd use 2,3-BPG phosphatase and detect the phosphate released, either photometrically by the molybdenum blue reaction, or fluorometrically (doi:10.1021/bi00193a013). The latter method is more expensive, but also more sensitive. An even more sensitive assay would be to detect the radioactivity released from 2-[32P]-BPG (2-[33P]-BPG) by extraction into organic solvent (https://www.jbc.org/content/244/23/6493.short). Instead of the benzene recommended in that paper I'd use ethyl acetate, however, works just as well and is a lot less toxic.
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