A. Gel filtration

B. Affinity chromatography

C. Cation exchange

D. Anion exchange

Correct Answer: B. Affinity chromatography

Affinity chromatography exploits specific, high-affinity binding between a protein and its substrate (ligand) immobilized on the stationary phase, making it ideal for purifying proteins with strong substrate interactions.

Option Analysis

A. Gel filtration
Size exclusion chromatography separates by molecular size/hydrodynamic radius through porous beads. Proteins binding substrates don’t separate differently from non-binding proteins of similar size.

B. Affinity chromatography (Correct)
Ligand (substrate analog) covalently attached to matrix specifically captures target protein via reversible biological interaction. Washing removes impurities; elution disrupts binding (pH change, competitor). Achieves >95% purity in one step.

C. Cation exchange
Separates by net positive charge at specific pH. Substrate binding doesn’t correlate with charge differences between proteins.

D. Anion exchange
Separates by net negative charge. Like cation exchange, ignores substrate specificity.

Affinity chromatography is best to separate a protein that binds strongly to its substrate through specific ligand-protein interactions—gold standard for enzyme purification in GATE Life Sciences.

Binding Mechanism

Substrate analog immobilized on agarose beads captures target enzyme via lock-and-key interaction. Non-specific proteins wash through; elution uses competitive ligand or pH shift.

Technique Comparison

GATE Applications

Essential for protein purification questions in biochemistry. Used for His-tag, GST-fusion, antibody purification in molecular biology research.