Materials: Reaction Buffer 0.5 M Tris HCl pH 8.0 with 5 mM MgCl2 Alkaline phosphatase: 500...

Materials:

Reaction Buffer 0.5 M Tris HCl pH 8.0 with 5 mM MgCl2

Alkaline phosphatase: 500 μg/mL stock solution in the buffer above. Keep enzyme on ice!

p-nitrophenyl phosphate: 1000 μM and 10000 μM stock solutions in reaction buffer

1) Using the enzyme stock solution above and the dilution equation, calculate the volumes needed to prepare 2 mL of three enzyme dilutions consisting of 100 μg/mL, 125 μg/mL, and 150 μg/mL. Use the reaction buffer to make these dilutions.

2) Calculate the amount of substrate needed for each 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 solution concentrations listed above. Use the 1000 μM stock solution to calculate dilutions from 10-200 μM and the 10,000 μM stock solution to calculate from 500 μM-2000 μM. The final volume is 1.0 mL (or 1000 μL) for each dilution. Also calculate the volume of buffer needed, given that we will add 100 μL of the enzyme solution and the final volume is 1.0 mL (or 1000 μL) for each dilution.

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Expert Solution

queen_honey_blossom answered 1 year ago

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