Materials:
Reaction Buffer 0.5 M Tris HCl pH 8.0 with 5 mM MgCl2
Alkaline phosphatase: 500 μg/mL stock solution in the bufferabove. Keep enzyme on ice!
p-nitrophenyl phosphate: 1000 μM and 10000 μM stock solutions inreaction buffer
1) Using the enzyme stock solution above and the dilutionequation, calculate the volumes needed to prepare 2 mL ofthree enzyme dilutions consisting of 100 μg/mL, 125 μg/mL,and 150 μg/mL. Use the reaction buffer to makethese dilutions.
2) Calculate the amount of substrate needed foreach of the following concentrations 10 μM, 25 μM, 50 μM, 100 μM,200 μM, 500 μM, 1000 μM, and 2000 μM using the two stock solutionconcentrations listed above. Use the 1000 μM stock solution tocalculate dilutions from 10-200 μM and the 10,000 μM stock solutionto calculate from 500 μM-2000 μM. The final volume is 1.0 mL (or1000 μL) for each dilution. Also calculate the volume ofbuffer needed, given that we will add 100 μL of the enzymesolution and the final volume is 1.0 mL (or 1000 μL) for eachdilution.
