I would like to performe the GRIESS reagent kit from biotium. The nitrate reductase enzyme (Sigma catalog no. N7265) has an activity ≥300 U/g. By protocol I am to use it at a final concentration of 300 U/L. The enzyme is in lyophilized powder form. How many microliters of grade water should I add? And how many microliters should I take of my solution to have final concentration of 300 U/L?
Please provide your suggestions.
The actual specific activity may be written on the container, or it may be available if you enter the lot number into the product web page to get the certificate of analysis.
It's usually a good idea to dissolve enzymes to at least 1 mg/mL for storage as a stock solution, if there is a sufficient amount. The solvent should either be water or a buffer, depending on what the enzyme was in when it was lyophilized. If it was lyophilized from a buffer, you should dissolve it in water, preferably to the same protein concentration it was when it was lyophilized so that the buffer will be the same. If it was in water when it was lyophilized, then you should dissolve it in a suitable buffer to support its stability.
Let's suppose, as an example, that the actual specific activity is 400 U/g (=0.4 U/mg) and you purchased 10 units. The amount in mg would be (10 U)/(0.4 U/mg) = 25 mg.
If you have an analytical balance capable of weighing at the 1 mg or even 10 mg level, you would not have to dissolve all of the solid at once. Suppose you weigh out 10 mg. If you dissolve it in 1 mL of solvent (water or buffer), the concentration (in the example) would be (10 mg/mL)x(0.4 U/mg) = 4 U/mL = 4000 U/L. Since you want 300 U/L, you would dilute this solution into the final reaction in a ration of 300:4000 = 3/40.
The product appears to be sold in units, in which case the u/g value has no bearing on what you need to do. The largest pack is 10 units, and you want 300U/L final conc: or 0.3U/ml. Your reconstituted stock therefore must be >0.3 U/ml if you are going to add it other reagents and still achieve a final concentration of 0.3U/ml. For example, you could reconstitute the 10U of enzyme in 333ul, which is 30U/ml i.e. 100x stock. Then you would add 1ul to 99ul to achieve the required conc.
The last sentence of your question however is unclear, as the concentration of enzyme once reconstituted won’t change regardless of how many uL you withdraw.
To begin with the N7265 does have lot specific CofA associated with it, so you will more likely find the exact Activity level. For example the most current lot for N7265 is 481 Units/g which is as well 0.481 Units/mg. For this example if you want 300 Units/L, which is 0.3 Units/ml, you can for example take a 2 units vial and dissolve in 6.66 ml of your desired diluent and you will be at 0.3 Units/ml or 300 Units/L.
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