When I feed DHZ (a plant hormone cytokinin) to pea plants, I observe a novel peak on HPLC. I don't know what it is and I'm planning on identifying the compound, but in the meantime I'd like to proceed with measuring the conversion in vitro, because then I could produce more of the compound more easily for the identification.
So far, I tried to homogenize the plant material and add about 1 : 1 buffer (200 mM K2HPO4 titrated by citric acid to pH 4 or 7; 0.1% Triton X-100; 5 mM DTT; radioactive DHZ). I haven't cleared the extract prior to the activity measurement to make sure I don't lose the activity and cofactors, but even after prolonged incubation, I haven't observed the peak.
How could I proceed? I'm not much keen to try various buffers and additives (pH; salt; detergent; reducing agent) and cofactors (like anything), because that would be so many options and I have no idea what should I try.
I'm not much in favour of waiting for identification of the product either for two reasons
1) that will take time
2) even when I will identify it, I doubt I will guess the cofactors
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