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Biological Sciences 102
Score (200): ______________
(10 pts.) Ammonium sulfate cuts are often done early in a protein purification preparation.
How many ammonium sulfate cuts would you typically recommend, when doing an enzyme purification: one,
two, or three? Defend your answer.
(10 pts.) Preparative isoelectric focusing gels require several components, besides water and a power
supply, to work. What are each of those components and what is each doing?
A sizing column is a very gentle way of separating, i.e. purifying, proteins. Let’s imagine that you are
using a sizing column to separate your enzyme Z from contaminating proteins A, B, C, and D.
(10 pts.) a) Let’s say that proteins A, B, C, and D have molecular weights of 10,000; 50,000; 100,000; and
30,000; respectively. In addition, each of these proteins is a dimer. What would the order of elution be for these
four proteins (A, B, C, and D) coming out of the column? Explain.
(10 pts.) b) When we assay all of the fractions of the above sizing column for our enzyme Z, using our enzyme
Z assay, we do not find it! This is initially very worrisome. However, we remember that sizing columns are
very gentle and hence denaturation of our enzyme Z structure is highly unlikely. Instead, it is possible that
something was “lost” during the sizing column elution. When that something is found, and put back with our