Question

A protein retained on an affinity chromatography column is usually eluted off the column by (circle the correct answer) A. increasing the salt concentration in the buffer B. changing the temperature of the elution buffer. C. allowing the retained protein to naturally come off the column after the non-specifically bound proteins have first passed through the resin. D. adding the protein's free ligand.

Answer 1

Affinity chromatography is a technique for separation of ionizing molecules based on total charge. This technique is used to separation of similar types of molecules, which are difficult to separate by other techniques. Affinity chromatography separates these molecules by altering the pH of the buffer. It is used to separate proteins, peptides, amino acids, and nucleotides. Amino acids present in proteins or peptides are zwitterions compound that have both positively charged amino and negatively charged carboxyl groups. Depending on the pH of the environment, the proteins carry positive, negative or no charge. The pH at which the molecule has no charge is the isoelectric point or Ip. This isoelectric point value is determined by primary sequence of the protein.

The protein has a net negative charge in a buffer with a pH greater than the pI of the protein. A positively charged anion exchange resin is used to separate this protein. Similarly a negatively charge cation exchange resin is chosen, when the protein has a net positive charge. This protein has a positive charge, as it is placed in a buffer with a pH lower than the pI of the protein of interest.

Positively charged proteins bind to a negatively charged anion exchanger.

The column is washed after all appropriately charged protein bind the resin. In order to elute the proteins, salt concentration of the buffer is varied. There are functional groups on certain amino acids that are charged. The charged salt ions compete with bound proteins for the charged resin functional groups. Low salt concentration will elute proteins with few charged groups. On the other hand, a high salt concentration is used to elute proteins with more functional groups. pH alteration can also done to elute the proteins, but it’s less commonly used.

Right option is A.