Which of the following statement about isoelectric focusing is correct

(a) Proteins separated by isoelectric focusing cannot be tested for biological activity
(b) Proteins separated by it can be tested for biological activity
(c) The separation of proteins is only based on charge
(d) The separation of protein is only based on size

✅ Correct answer: (C) The separation of proteins is only based on charge

Explanation:

Isoelectric Focusing (IEF) is a technique that separates proteins based on their isoelectric point (pI), which is the pH at which a protein has no net charge and stops moving in an electric field.

• Proteins migrate in a pH gradient until they reach their pI, where they have zero net charge and stop.
• The separation is entirely based on charge, not size.
• IEF is used in 2D-PAGE as the first dimension to separate proteins by charge before SDS-PAGE separates them by size.

Why Not the Other Options?

• (A) Proteins separated by isoelectric focusing cannot be tested for biological activity ❌

  • Incorrect, because IEF does not denature proteins as SDS-PAGE does.
  • If mild conditions are used, proteins can retain biological activity.

• (B) Proteins separated by it can be tested for biological activity ❌

  • Partially true, but not always.
  • If denaturing conditions (e.g., urea) are used, proteins may lose activity.
  • However, native IEF can preserve function, making this statement not universally correct.

• (D) The separation of protein is only based on size ❌

  • Incorrect, because IEF does not separate proteins by size, only by charge (pI).
  • SDS-PAGE separates by size, not IEF.

Final Answer:

✅ (C) The separation of proteins is only based on charge