Affinity chromatography fundamentals: What is the core principle by which affinity chromatography separates biomolecules?

Introduction / Context:
Affinity chromatography exploits natural or engineered high-affinity interactions (for example, enzyme–substrate analog, antibody–antigen, His-tag–Ni2+) to purify a specific protein from complex mixtures with high selectivity.

Given Data / Assumptions:

• A stationary phase presents immobilized ligand (for example, Ni-NTA, Protein A, biotin, substrate analog).
• Target protein possesses a complementary binding site or tag.
• Elution occurs by competitive ligand, pH, or ionic strength change.

Concept / Approach:
Only biomolecules with the correct binding epitope are retained strongly. Non-specific binders are washed away under conditions that preserve the specific interaction. Elution disrupts the specific interaction while maintaining protein integrity.

Step-by-Step Solution:
Bind: load clarified lysate onto ligand-bearing resin.Wash: remove weak/non-specific binders.Elute: add competitor or change pH/salt to release target.

Verification / Alternative check:
Single major band by SDS-PAGE after elution indicates specificity; activity assays confirm functional recovery.

Why Other Options Are Wrong:

• Aggregation/precipitation: Not the mechanism of affinity capture.
• Random silica adsorption/hydrophobic forces: Describe other chromatographies (HIC, reversed-phase), not affinity.
• Gas–liquid partitioning: Pertains to GC, irrelevant to proteins in water.

Common Pitfalls:
Overloading columns, using incompatible buffers that block binding (for example, imidazole during Ni-NTA loading).

Final Answer:
Specific binding of a protein to an immobilized ligand (high-affinity interaction).