Protein Purification

1. Determine if the following statements are TRUE or FALSE. If the statement is FALSE
provide and explanation as to why.
a. Protein X precipitates at a ammonium sulfate concentration fo 2 M. To purify
protein X from a mixture of proteins it is best to carry out one precipitation
with 2M ammonium sulfate.
b. In SDS-PAGE the addition of SDS (sodiumdodecylsulfate) denatures the
protein and coats the protein with negative charge.
c. In ion exchange chromatography proteins are separated based on their
d. In gel filtration chromatography proteins are separated based on their net
e. You purify a protein Y from a mixture of proteins and perform SDS-PAGE to
determine purity. You observe one band in the lane containing the purified
protein at the expected molecular weight. Based on this SDS-PAGE result
you can be sure that you have purified the correct protein.
f. In anion exchange chromatography the chromatography matrix will be
negatively charged.
g. In SDS-PAGE protein with a larger molecular weight will migrate less and
proteins with a smaller molecular weight will migrate further.
h. In size-exclusion chromatography the proteins with a smaller molecular
weight will elute first.
i. Dialysis works on the principle of molecular weight.
j. Western blots and protein mass spectrometry can be used to confirm the
identity of proteins.
2. Consider two proteins mixed together in a buffer at pH 6.0. If protein A has a pI of
4.5 and protein B has a pI of 7.7, which one (or both or neither) will be retired by an
anion exchange matrix?
3. You perform gel filtration chromatography on a protein mixture and identify that
your protein elutes with an estimated molecular mass of 50 kDa. You take the
fractions that contain the protein of interest and run SDS-PAGE. The SDS-PAG has
Protein Purification
a single band at 25 kDa. Provide a plausible explanation to reconcile the difference
in molecular mass obtained by gel filtration and SDS-PAGE.
4. You joined anew biotechnology company and have been tasked with purifying a
protein from human pancreatic tissue. Provide a well thought out description of
how you might purify your protein. Assume that your protein is an enzyme and you
have an assay to detect the presence of your protein. Your description should
contain how you would check for the purity of the protein as well as its identity.

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