451A Assignment - 2
A graduate student was studying three different proteins X, Y and Z. He accidently mixed all
three of them. Unfortunately he did not have any more stocks of these proteins in the lab.
He/she thought of telling his mistake to his instructor but was afraid to disclose it. He/she
thought about the nature of all three proteins and designed a strategy to separate them by
simple biochemical techniques. Protein X is 167KDa, Y is 15KDa and Z is 13KDa. The
student also knows that protein Y is a basic protein and protein Z has three FLAG epitope tag
at its C-terminus. FLAG epitope consists of 8 amino acids and is used commonly to tag
proteins for immunopurification.
1. Based on the above information, what method(s) should the student follow to separate
X, Y and Z? Include in your answers different kinds and properties of resins the
student should use.
2. Once purified, how would you check the quality and quantity of the purified proteins,
and what should be the approach?
3. He also ran purified proteins on a non-denaturing gel. The result is shown below (left
panel). Explain the gel and result. He than heated all three proteins. The result he
observed is as shown on the gel (right panel). What do you think has happened to
protein Z. Explain two different results obtained before and after heating protein Z.
X
Y
X
Z
Y
Z
Heat proteins at
90C for 3mins before
loading
4. He is interested in separating different forms of protein Z. What an analytical
approach would you suggest to separated them and determine their molecular weight.
5. Explain why the molecular mass of fibrinogen is significantly overestimated when
measured using a calibrated gel filtration column (see figure 6-10 in Voet & Voet), but can
be determined with reasonable accuracy from its electrophoretic mobility on an SDSpolyacrylamide gel (see Table 6-5).