I'm measuring ADP ribose cyclase from fresh tissue homogenate using fluorescent detector ( microplane reader ). The graph shows the fluorescent of the product high at 0 mins, then decrease RFU, then at 15 min start going up so from 15 -60 min it looks like a enzyme reaction graph. Can Michealis menten equation work with this ? Cause I really don't know when the real 0 mins
In principle, the answer to your question would be yes, but in practice most likely it is no.
I mean, you've got so many possible reactions going on in a tissue homogenate, that you can hardly trust the readout of an assay: your substrate may be consumed, your product depleted or converted into something else, or generated through a different pathway...
As the late Arthur Kornberg would put it "Never waste clean thinking on a dirty enzyme".
Maybe. You are right by saying fluorecence, but this might not be the case. (In terms of kinetics, maybe the second?) Does it work with an enzyme? Can the product specify resonance by time/ decay and product?
Alessio Peracchi I agree with you that the homogenate got so many reactions outside of my aimed reaction. However this is the fluorescence assay ( only the fluorescence coming out from the product is detected ) so I think it won't be the problem of deciding whether it's the right reaction or not. The increasing fluorescence will show that there is reaction. But I'm very critical about when the reaction starts. Because as you said, there are so many things in the fresh tissues (cells/ membrane/ etc) so the substrate might take time to get to the enzyme.
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