I am optimizing pH for my cellulase enzyme with substrate CMC and pNPG. Can anyone provide me with a good protocol for pH optimization?
Im not sure how to do it with CMC, but for pNPG is quite easy because when you hydrolyzed pNP, you get yellow color.
You need a plate reader to measure changes in absorbance at 404 nm.
This is the protocol: The optimal pH was determined using 100 mM phosphate-citrate buffer (pH 3 to 7) and 50 mM glycine-NaOH buffer (pH 7.5 to 9.5) in the presence of each purified enzyme (1 μg in 100 μl) and 2 mM pNP-G. After do this you will have a curve for each pH, just analyze the slope, thats all. If you use diferent concentration of pNPG you can also calculate Km and Vmax.
Regards,
Gonzalo
Thanks alot Gonzalo, I am just confused what is the buffer in which i should resuspend my protein, since that buffer has to maintain the pH of Phosphate-citrate that i will add in each reaction that will vary pH range 3-7
When I resuspend for the first time one enzyme, I do it in 100 mM HEPES pH 7 or Tris 100mM pH 7. Then when you know the optimal pH you can do it in another if you want.
Anyway, if your confution its about the reaccion, doesnt matter the buffer where do you resuspend, because the reaction is in 100ul (and has its own buffer), and you only put 1 ul of your enzyme, the buffer of the enzyme wont change the pH of the reaction.
Regards,
Gonzalo
Since I am optimizing the reaction, so i have my enzyme activity very little. I use 80ul to have activity. thats why i am more worried about the buffer of the enzyme.
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