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activity assay optimization - (Jul/03/2018 )

I need some help with enzyme activity assay optimization. It's a recombinant human enzyme, here's the assay description


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:


a) keep the concentration of donor and acceptor constant and increase the amount of enzyme until the reaction rate will reach a plateau, like in the pictureAttached Image


it would mean that at a certain concentration (x) there's not enough substrates in the reaction. So, the next step would be:


b) 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!




within detectable limits of malachite green, you should perform a substrate saturation assay series (curve) using a fixed amount of enzyme. although, the kinetics of the enzyme are probably well known.


Are there any guidelines how to choose the amount of the enzyme, or is it simply trial and error?  


Also, my goal is not to study the kinetics of the enzyme, but to demonstrate that it can be recombinantly expressed in an active form (and compare its activity with the commercially available enzyme). 


then, you want to use enough to give you a reasonable response with your detection method. you also want to match the purity and quantity of the commercial enzyme. if you can't match purity then you should match units of enzyme activity in the assay.


Thanks, mdfenko!