13. For a laboratory project you asked to purify the enzyme lactate dehydrogenas

Q: 13. For a laboratory project you asked to purify the enzyme lactate dehydrogenas

amount of CAPS buffer 25mM = wt*1000 / 221.3*200 Wt = 1.1065 gm. amount of MgCl2 5mM =Wt*1000 / 95.2*200 Wt = 95.2 mg amount of Triton X-100 100 x V1 = 0.2 x 200 ml V1 = 0.4 ml. amount of pepstatin A 25000 ug/ml x V1 = 10 ug/ml x 200 ml V1 = 2/25 = 0.08 ml or 80 ul from stock solution.

ID: 276616 • Letter: 1

Question

13. For a laboratory project you asked to purify the enzyme lactate dehydrogenase (LDH) The first step of this project is to prepare the lysis buffer that you will use to extract the enzyme The protocol you are following says the LDH lysis buffer should contain: 25 mM CAPS (a pH buffer) at pH 10 5 mM MgCl2 0.2% Triton X-100 (a detergent) 10 ug/ml pepstatin A (a protease inhibitor) You find the following reagents on the shelves in lab a bottle of CAPS powder; MW is 221.3, pKa is 10.4 a bottle of MgCl2 crystals; MW is 95.2 a bottle of Triton X-100 (this detergent is a liquid in its pure state) and a stock solution of 25 mg/ml pepstatin A You need to make 200 ml of this buffer. Tell how you would make this solution if you were to

Explanation / Answer

amount of CAPS buffer

25mM = wt*1000 / 221.3*200

Wt = 1.1065 gm.

amount of MgCl2

5mM =Wt*1000 / 95.2*200

Wt = 95.2 mg

amount of Triton X-100

100 x V1 = 0.2 x 200 ml

V1 = 0.4 ml.

amount of pepstatin A

25000 ug/ml x V1 = 10 ug/ml x 200 ml

V1 = 2/25 = 0.08 ml or 80 ul from stock solution.

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13. For a laboratory project you asked to purify the enzyme lactate dehydrogenas

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