I'm preparing enzyme-responsive polymeric nanosystems and according to the manufacturer's information, the enzyme is in
-» "50 mM sodium acetate buffer, 1 mM EDTA, pH 5.0.";
-»"≥10 units/mg protein" and
-» unit definition "One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol of compound x per min at 40°C, using 100 mM Na+/K+ pH 6.0, with 1.33 mM EDTA and 2 mM DTT as the activation buffer."
The flask has 50 micrograms of enzyme.
Concentration: 0.451 mg/mL.
416.0 U/mgP
I wanted to prepare 0.5 UN/ mL solution.
Thanks very much
Hi there,
If the stock 416U/mg of protein and 0.451mg/mL then it's also 416/0.451= 922.4U/mL. So the dilution factor to apply will be 922.4/0.5= 1845.
Depending on the required volume for the dilution, you may consider the basic operation: putting 1µL of the stock into 1844µL of diluent.
Assuming that the specific activity is indeed 10 U/mg and the protein concentration 0.451 mg/mL, then the solution is 10 x 0.451 (U mg)/(mg mL) = 4.51 U/mL. Thus, you need a 9.02-fold dilution (1 mL made to 9.02 mL final volume with a suitable buffer). Note, however, that the manufacturer specified only a minimum activity, the actual activity may be higher and should be checked.
A good book to learn this type of calculations from is I.H. Segel: Biochemical calculations: how to solve mathematical problems in general biochemistry, New York (Wiley) 1976, ISBN 0-471-77421-9. Maths hasn't changed over the last 50 years, so the book is still useful. The chapter on enzyme kinetics in my own "Fundamentals of protein structure and function" may also be helpful.
Btw, the enzyme unit U (1 U = 1 µmol/min) is outdated and doesn't fit into the SI. Instead, you should be using the katal (1 kat = 1 mol/s, 1 U = 16.7 nkat).
Hi Miguel,
if the actual activity of your protein is 416 U/mg (and not just 10 U/mg) and the concentration of the protein is 0.451 mg/mL, then you need to multiply 416 by 0.451= 187.6 U/mL. From that, if you need a solution at 0.5 U/mL the dilution factor will be 187.6x2= 375.2 (i.e. 1 µl of the solution in 374.2 µl of the reaction)
@Marco: you are definitly right! I made a slight calculation error... ;)
- Badges
- Science method