You have a mixture of the following proteins Protein A 25 kD

You have a mixture of the following proteins: Protein A, 25 kDa, pl 7.5 Protein B, 100 kDa, pl 5.2 Protein C, 25 kDa, pl 9.5 Protein D, 45 kDa, pl 5.2 Protein E, fused with an MBP tag, 100 kDa, pl 9.0 You carry out the following experiments. You run the mixture over an ion exchange column in a low salt buffer at pH 7.0. You wash with a low salt buffer, elute with an increasing salt gradient. and see the elution profile shown However, you forgot to write down the column used. Oops! When you run SDS-PAGE of the fractions corresponding to each peak, you see the gel on the right. Using all of this information: A) what kind of ion exchange column must you have used to generate the results shown? Please explain your reasoning in detail. B) Please label the name and molecular weight of each protein next to the corresponding band in the gel above. C) What kind of column do you need to separate two proteins in peak 1? Please explain your reasoning in detail.

Solution

Answer:

A. The column used is a cation exchanger.

Ion exchange protein purification is possible because most proteins bear non zero net electrostatic charges at all pHs except at pH = pI (isoelectric point). At a pH > pI of a given protein, that protein becomes negatively charged (an anion), at the pH < pI of that same protein, it becomes positively charged (a cation).

So, proteins B and D becomes negatively charged at pH 7 and elutes first since the binding is not efficient due to similar charges in the ion exchange beads.

Protein A elute second since it is almost neutral at pH 7.

Proteins C and E are positively charged and binds to the column efficiently. So, elutes last.

B. Peak 1: Migrated less: Protein B (100 kDa)

Migrated more: Protein D (45 kDa)

Peak 2: Migrated less: Protein A (25 kDa)

Peak 3: Migrated less: Protein E (100 kDa)

Migrated more: Protein C (25 kDa)

C. The proteins in peak 1, i.e, B and D are 100 kDa and 45 kDa respectively. They can be separated efficiently using a size eclusion chromatography (FPLC) column.