Q.40 Purification of 6×His-tagged protein using Ni-NTA column is an example
of ________
(A) affinity chromatography
(B) hydrophobic-interaction chromatography
(C) ion-exchange chromatography
(D) size-exclusion chromatography
Purification of 6×His-tagged protein using Ni-NTA column is an example of affinity chromatography.
This technique leverages the specific binding between the histidine tag and nickel ions on the resin for highly selective protein purification. The correct answer is (A) affinity chromatography.
Option Analysis
Affinity Chromatography (A): Proteins with a 6×His-tag bind specifically to Ni-NTA resin through coordination between histidine imidazole groups and Ni²⁺ ions, allowing impurities to wash away before elution with imidazole. This method achieves high purity in one step under native or denaturing conditions.
Hydrophobic-Interaction Chromatography (B): Separates proteins by surface hydrophobicity using high-salt buffers to promote binding to hydrophobic ligands, followed by low-salt elution; unrelated to His-tag specificity.
Ion-Exchange Chromatography (C): Relies on protein net charge interactions with oppositely charged resin, modulated by pH and salt gradients; no role for metal chelation.
Size-Exclusion Chromatography (D): Separates by molecular size through porous beads, with larger proteins eluting first without binding; ignores tag-based affinity.
Purification of 6×His-tagged protein using Ni-NTA column represents a cornerstone of recombinant protein isolation in molecular biology. This affinity-based method enables rapid, high-purity recovery from complex lysates, vital for biotechnology research and CSIR NET exam preparation.
Core Principle
The 6×His-tag (six histidine residues) forms coordinate bonds with Ni²⁺ immobilized on NTA resin, exhibiting high specificity (K_d ~10-13 M). Cell lysates bind selectively during loading, followed by washes to remove contaminants and imidazole elution to disrupt interactions. Yields often exceed 90% purity in single steps.
Procedure Steps
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Equilibration: Ni-NTA column with lysis buffer (50 mM phosphate, 300 mM NaCl, 10-20 mM imidazole, pH 8.0).
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Binding: Load cleared lysate; His-tagged protein captures via Ni²⁺ chelation.
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Washing: 20-50 mM imidazole removes weak binders.
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Elution: 250-500 mM imidazole releases target protein.
Comparisons with Alternatives
| Technique | Basis | Ni-NTA Relevance |
|---|---|---|
| Affinity (Ni-NTA) | Specific ligand-tag binding | Direct match for 6×His |
| Hydrophobic | Surface hydrophobicity, high-salt bind | No His-tag use |
| Ion-Exchange | Net charge differences | Charge-based, non-specific |
| Size-Exclusion | Molecular size via pores | Size sorting only |
Ni-NTA excels in specificity over charge or size methods.
CSIR NET Applications
Ideal for exam questions on protein purification techniques, emphasizing His-tag affinity over other chromatography modes. Combines with SDS-PAGE for purity verification.
