Just need answer for part a) You are given 0.1 kg of beef liver. Your goal is to
ID: 1073458 • Letter: J
Question
Just need answer for part a)
You are given 0.1 kg of beef liver. Your goal is to purify the Alcohol Dehydrogenase (ADH). You homogenize the liver in 100 ml of pH 7.8 buffer. After centrifugation, the supernatant volume is 75 ml. You make 100 mu l of a 200-fold dilution of the supernatant, and add it to 2ml of .Coomassje. reagent. The resulting absorbance at 595 nm is 0.37. At the same time, you add 100 mu l of a 0.25 mg/ml solution of a standard protein to 2 ml of Coomassie reagent. The resulting absorbance at 595 nm is 0.42 What is the protein concentration in the supernatant? You perform an ADH activity assay on the supernatant fraction, anion exchange fraction and the gel filtration fraction. For all assays, you add 5 mu l of the fraction to a final volume of 1 ml of assay mixExplanation / Answer
concentration of standard protein in 2 ml reagent = 0.25 x 0.1
= 0.025 mg
absorbance = 0.42
let Cx be the concentration of protein in dilute solution
then,
Cx/(Cx + 0.025) = 0.37/0.42
0.42 Cx = 0.37 Cx + 9.25 x 10^-3
Cx = 9.25 c 10^-3/0.05
= 0.185 mg
So concentration of protein in 2.1 ml solution = 0.185 x 2.1/0.1
= 3.885 mg
a) Concentration of protein in original solution in original solution = 3.885/75
= 0.052 mg/ml
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