Separation Principle Based on Isoelectric Point (pI)

The two monomeric proteins have identical molecular weights but differ in their isoelectric points (pIs): 5.6 and 6.8. The isoelectric point is the specific pH at which a protein carries no net electrical charge. Differences in pI indicate differences in the net charge of the proteins at any given pH other than their respective pIs.

Evaluating Separation Techniques

• Denaturing Polyacrylamide Gel Electrophoresis (PAGE): While PAGE separates based on size and charge, the denaturing conditions disrupt the native charge properties crucial for separation based on pI differences. Since the molecular weights are identical, separation based on size is also impossible.
• Size-Exclusion Chromatography (SEC): This technique separates molecules based solely on their size or hydrodynamic volume. As both proteins have identical molecular weights, SEC cannot differentiate between them.
• Ion-Exchange Chromatography (IEC): IEC separates molecules based on their net surface charge at a specific buffer pH. Proteins with different pIs will exhibit different net charges at a chosen pH. By selecting a pH where the two proteins have distinct charges (either both charged but differently, or one charged and one neutral/oppositely charged), they can be separated on an appropriate ion-exchange column (anion or cation exchanger).
• Nickel Affinity Chromatography: This method targets the His-tag. Since both proteins are His-tagged and have the same molecular weight, they are likely to bind similarly to the nickel column, making separation based on this technique alone difficult without additional information suggesting differential binding affinities.

Conclusion on Protein Separation

Ion-exchange chromatography is the most suitable technique because it directly exploits the difference in the proteins' net charge, which is determined by their distinct pIs (5.6 and 6.8). By adjusting the buffer pH, a charge difference can be established, enabling separation.