Need to perform in vitro reaction with substrates and purified protein (Methyl esterase/alpha/beta hydrolase super family)
1. What are the essential cofactors?
2. Which buffer solution/pH should I use for the reaction?
Please share your experiences
Thanks
Buffer solution and pH is something you have to try, every enzyme behaves differently. I usually use the one given in the literature if it is a known enzyme, and for fun I try PIPES, MOPS and Tris since they also have different pH buffering ranges to try for optimal pH (some researchers don't like this approach because of the differences in ionic strength).
Regarding cofactors, if you are lucky your protein will be coexpressed with its cofactor. Blast it against conserved domains (CDD) and this will tell you what binding domains to cofactors there are, I guess this is usually FMN.
Also you should test optimum temperature and if activity decreases when EDTA is present your enzyme requires a metal cofactor.
Good luck!
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