My research focus on the production of xylanase, and pectinase in SSF condition. I wanted to do enzyme assay of the crude enzyme extracted using DNS method.
My question is, for each sample do I have to make enzyme blank or buffer blank?
I'm thinking of doing enzyme blank since there is possibilities of existence of reducing sugar that was extracted along during crude enzyme extraction. But it would be tedious and time consuming because I have to prepare different enzyme blank for each different sample.
or I can do buffer blank which is a lot easier.
For the cellulolytic enzyme assays, it is recommended to use reagent, substrate and enzyme blanks for each samples to nullify the background effects from substrate containing enzyme samples. Here reagent and substrate control (triplicates) will remain same for the all the samples but you have to take different enzyme control for each enzyme samples.
You are a scientist.... You do ALL the possible controls ...then evaluate them... Please don't look for shortcuts.
A hard-learned lesson: run as many blanks as you can think of. It's the blank (or other control) that you do not run, for which you will eventually regret not having the data. Buffer, substrate, enzyme, and even a totally null blank are all appropriate in most situations.
there are No easier ways to do enzyme assays the results we produce should be reproducible and has to be validated so follow the well accepted procedures
I'm agree with Craig A Gelfand and Robert L Baxter you should have all possible controls done. If not you take the risk to regret and you will have to run again the experiments.
For measuring enzyme activities, controls are must and should. You need to have Test (main reaction), Control (enzyme blank), and buffer blank for calculation of enzyme activities. My experience: Test values obviously change, but control values also will change as you do the time course in SSF or SmF
Check this one, this will tell you how to do enzyme assay.
Ghose TK (1987) Measurement of cellulase activities. Pure Appl. Chem. 59:257–268.
It is necessary to do all experimental controls although be expensive in time and reactive. It is the only way to put on evidence and approach what is happening there and also to show very convincing results. You can and must work very hard, and even so, never reach the truth. The truth is unattainable. You use all the methods at your disposition. Confirm and to verify. The recomendations of the rest of colleagues are very important and useful..
I'm agree with Craig and Robert that you have all possible controls and after analyse it.
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