Question

what is charge of the cationic exchange beads?

which protein comes off the ion exchange column first?( Use the charge on the protein?

Why does the second protein come off with 2M NaCl? Is there another possible way of removing the second protein from the column?

Both of the proteins used here are colored. How could we separate proteins that were colorless or white

Where is myoglobin found? what is its function?

Explain how ion-exchange chromatography works?

Why might you use affinity chromatography?

Biochemistry

Answer 1

Cationic exchange beads are negatively charged and help in the exchange of cations.

The protein having higher negative charge will come off the ion exchange column first.

Myoglobin is an oxygen binding protein containing iron. It is found in muscular tissue of most of the vertibrates and in almst all mammals. The function of myoglobin is similar to that of haemoglobin i.e. myoglobin helps in the transport of oxygen during glucose metabolism. The affinity of myoglobin towards oxygen is more than that of haemoglobin.

Ion-exchange chromatography: ion-exchange chromatography is used to purify proteins and other charged ions. In  ion-exchange chromatography, the surface of a tube is coated with positively charged immobilized cation. These immobilized cations have affinity towards negatively charged anions. Now when a solution containing both positively charged and negatively ions/groups pass through the tube, the anions are attached to the surface containing immobilized cation where as the cations are flown through the solution(mobile phase). In this way the positively and negatively charged ions are separated. The pH of the solution(mobile phase) must lie in between the pKa of the charged group in the solution and the pKa of the charged group on the solid surface.

Affinity chromatography: It is used to separate biochemical mixtures based on highly specific interactions like antigen-antibody interaction, enzyme - subtrate interaction or receptor - ligand interaction.