Question

Your summer research project requires that you purify a novel protein expressed in bacteria. After centrifugation of your cell lysate, you find that there are 4 major proteins left in the supernatant fraction that have the following pI values: protein A = 8.0, protein B = 4.0, protein C = 6.8, and protein D = 5.3. If these proteins are passed over a cation exchange column in a solution buffered to pH 5.2, what will be the order in which they will pass through the column (from first to last to elute) if a linear gradient of increasing salt is applied? Why?

A, B, C, D

C, A, B, D

B, D, C, A

D, B, C, A

D, B, A, C

Answer 1

Answer.

The correct order is B,D,C,A.

Reason: Ion exchange chromatography is a process for separating proteins and other molecules in a solution based on differences in net charge. To ensure same, dissolve it in a buffer that is either above or below its isoelectric point depending on cation or anion column. For example, a protein with a pI of 7 bound to a cation column at pH 5 will elute by increasing the pH above 7. Also, more the difference is between pI and pH, the more effective protein gets eluted.

Here,

since in the given cation column, protein B and D have only pI less than pH, they will get eluted first. Comparing difference between pH and pI for B and D, B have larger gap with pH and will get eluted first.Protein A have higher gap than C and hence C will get eluted first.