Q.4 Size exclusion chromatography is used to separate the proteins on the basis of –
- Size
- Temperature
- Net binding specificity
- Net charge
Size exclusion chromatography separates proteins based on their size (hydrodynamic volume), using porous beads where larger proteins elute first. This makes “Size” the correct answer for this GATE Life Sciences question on protein purification techniques.
Question Solution
Correct Answer: Size. Size exclusion chromatography (SEC), also called gel filtration, purifies proteins by molecular size/shape without chemical interactions—larger molecules pass through column voids quickly, smaller ones enter pores and elute later.
Option Analysis
-
Size: Correct. SEC relies on hydrodynamic radius; proteins > pore size elute in void volume (first), while smaller ones delay in pores. Ideal for desalting, aggregate removal, and fractionation (10 kDa–10 MDa range).
-
Temperature: Incorrect. Temperature affects protein stability but not SEC mechanism; it’s run isothermally (4–25°C) to preserve native structure.
-
Net binding specificity: Incorrect. This describes affinity chromatography (e.g., Ni-NTA for His-tags), where ligands bind targets specifically.
-
Net charge: Incorrect. Ion-exchange chromatography separates by charge (anion/cation exchangers); SEC is non-interactive.
Introduction
Size exclusion chromatography is used to separate the proteins on the basis of size, a key technique in molecular biology and biochemistry for GATE Life Sciences students in Jaipur. This non-denaturing method purifies via porous matrices, essential for analyzing aggregates and oligomers.SEC Mechanism
Column beads (agarose/Sephadex) have defined pores. Sample flows; large proteins (e.g., aggregates) exclude from pores, elute at V0 (void volume, ~30% column). Small proteins permeate, elute at Vt (total volume). Resolution: Kav = (Ve – V0)/(Vt – V0), where Ve = elution volume.
No binding—avoids activity loss. Run time: 0.5–2 column volumes.
Comparison of Protein Separation Methods
Method Basis Stationary Phase Key Use Size Exclusion Size/shape Porous beads Desalting, aggregates Ion Exchange Net charge Charged resins Charge variants Affinity Binding specificity Ligands (e.g., antibodies) Tagged protein isolation Hydrophobic Interaction Hydrophobicity Alkyl chains Surface hydrophobicity SEC often polishes multi-step protocols (after affinity/ion-exchange).
Applications and Tips
Purifies monoclonal antibodies, estimates MW via standards. Tips: Load <5% column volume; use isocratic buffer (150 mM NaCl, pH 7). For GATE: Distinguish size (SEC) from charge (IEC)—common PYQ trap.
-
