Calculate the volumes of needed to make solutions of alkaline phosphatase and p-

Question

Calculate the volumes of needed to make solutions of alkaline phosphatase and p-nitrophenylphosphate with the given concentration and update the table below.

Reagents:

Reaction buffer: 0.5 M Tris HCl (pH 8.0) with 5 mM MgCl2

Alkaline phosphatase: 500 µg/mL stock solution in reaction buffer.

p-nitrophenylphosphate: 1000 µM and 10,000 µM stock solution in reaction buffer.

100 mM sodium phosphate pH 8.0.

100 mM D-phenylalanine.

100 mM L-phenylalanine

B. Substrate and inhibitor solutions (See lab handout Section C3, C-4 and C-5 and Section E for details.) [InhibitorNP(L)-Rbe(D)-Pbe Enzyme Buffer Total [SIHM Substrate (HL) (HL) (HL) (HL) (L 1000 0 5 1000 1000 3 3 1000 0 16 1000

Explanation / Answer

# [S] uM Substrate (uL) Inhibitor (uM) NaP (uL) L-Phe (uL) D-Phe (uL) Buffer (uL) 1 1000 100 0 800 2 100 100 0 800 3 25 25 0 875 4 1000 100 1 10 780 5 100 100 1 10 780 6 25 25 1 10 780 7 1000 100 0.5 5 785 8 100 100 0.5 5 785 9 25 25 0.5 5 785 10 1000 100 10 100 700 11 100 100 10 100 700 12 25 25 10 100 700 13 1000 100 5 50 750 14 100 100 5 50 750 15 25 25 5 50 750 16 1000 100 10 100 700 17 25 25 10 100 700

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