I need some help with enzyme activity assay optimization. It's a recombinant human enzyme, here's the assay description https://www.rndsyste...tein-cf_4950-gt
In short, the enzyme is incubated with donor and acceptor substrates, then the reaction is stopped by addition of Malachite Green reagents and enzyme activity is calculated.
I've tested a few combinations of donor/acceptor substrates and got some results, i. e. the enzyme is active. Now I need to optimize the assay so it would look good in a paper. I have some doubts about what to do:
it would mean that at a certain concentration (x) there's not enough substrates in the reaction. So, the next step would be:
use the same amount of the enzyme (x) in a few reactions with different (higher) concentrations of the substrates
c) increase the concentrations of the donor/acceptor substrates and use higher amounts of the enzyme (x, 2x, 3x, etc)
I have limited amounts of the essay reagents and since there are so many options I feel a bit lost about what to do...
Thanks in advance!