If your question is how to standardise the concentrations, then you may refer some literature for similar experiments done or theoretically choose concentrations of different components (enzyme, substrate, buffer, etc), and do the assay. If you do not get anticipated results, you may change parameters (concentration, pH, temperature, etc) one at a time, till you get maximum activity.
I always follow the KISS rule (keep it simple, stupid) and aim to have all stock solution at either 100X, 10X or 2X.
it would be various dependent on individual enzymes.
In general, the higher stock concentration, the better for enzymes, unless aggregation happens.
Substrate needs to be in a stock which is convenient for dilution and also avoid precipitate during dilution into aqueous buffer;
Total volume needs to optimized based on your labware: plate or vials. Smaller labware requires less reagents so it is ideal, but considering the accuracy, larger reaction volume would be better. Normally 96-well plate is very good. 384 -well plate is good for saving reagents and can be operated manually without relying on liquid handling system.
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