If a protein is placed in a buffer with pH higher than its pI and an electric field is applied, where will the protein migrate?

• A. An anion and migrate to the anode
• B. A cation and migrate to the cathode
• C. An anion and migrate to the cathode
• D. An uncharged particle and will not move

Answer: (A)

When the pH of the buffer is higher than the protein’s isoelectric point, the protein carries a net negative charge. This happens because deprotonation of acidic groups increases as pH rises, giving the molecule more negative charges. In electrophoresis, charged species migrate toward the electrode that attracts their charge: negatively charged particles move toward the positively charged anode, while positively charged particles move toward the negatively charged cathode. So a negatively charged protein will migrate toward the anode.

If the pH were below the pI, the protein would be positively charged and move toward the cathode; at the pI, the net charge is essentially zero and the molecule would not migrate significantly.