Q4 In affinity chromatography, the protein bound to the column can be eluted using a buffer containing
1. 0.1 mM NaCl
2. Acetonitrile
3. immobilized ligand
4. free ligand

 

Understanding Affinity Chromatography and Protein Elution

Affinity chromatography is a highly selective method used for protein purification based on specific interactions between a ligand and a target protein. It is widely used in molecular biology, biochemistry, and industrial biotechnology. Understanding how affinity chromatography works and how to elute the bound protein is essential for research and competitive exams like CSIR NET Life Science, IIT JAM, GATE Biotechnology, and DBT JRF.

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What is Affinity Chromatography?

Affinity chromatography is a technique that separates proteins or other biomolecules based on their specific binding affinity to a ligand. The ligand is immobilized on a stationary phase (usually a resin or matrix) and binds to the target protein through highly specific interactions such as:

• Antigen-antibody interactions
• Enzyme-substrate interactions
• Receptor-ligand interactions
• Protein-nucleic acid interactions

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How Affinity Chromatography Works

1. Binding Phase

• The sample mixture is passed through a column containing the immobilized ligand.
• The target protein binds specifically to the ligand, while other proteins and impurities pass through.

2. Washing Phase

• The column is washed with a buffer to remove unbound or weakly bound proteins.
• The target protein remains attached to the ligand due to its high affinity.

3. Elution Phase

• The bound protein is released from the ligand using a competitive agent or changing the buffer conditions.
• Common elution methods include:
  • Free ligand – The most effective elution method.
  • Change in pH or ionic strength.
  • Use of chaotropic agents or denaturing agents.

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Role of Free Ligand in Elution

The most effective way to elute a protein from an affinity chromatography column is by introducing a free ligand. The free ligand competes with the immobilized ligand for the binding site on the target protein, resulting in the release of the protein from the column.

Example:

• In enzyme-substrate affinity chromatography, the bound enzyme can be eluted by adding an excess of free substrate or a substrate analog.
• In antibody-antigen affinity chromatography, the bound antigen can be eluted using a solution containing the free antibody.

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Correct Answer to the Question

In affinity chromatography, the protein bound to the column can be eluted using a buffer containing:
✅ Correct Answer: 4. Free ligand

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Applications of Affinity Chromatography

Protein purification – Isolation of recombinant proteins from cell lysates.
Antibody purification – Purification of monoclonal and polyclonal antibodies.
Enzyme purification – Purification of enzymes and their inhibitors.
DNA-protein interactions – Studying DNA-binding proteins and transcription factors.

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Why Affinity Chromatography is Important for Competitive Exams

Affinity chromatography is a frequently tested topic in CSIR NET Life Science, IIT JAM, GATE Biotechnology, and DBT JRF. Candidates are expected to understand the mechanism, types of ligands, and elution strategies.

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Conclusion

Affinity chromatography is a highly effective method for purifying proteins based on their binding affinity to a ligand. The elution of bound proteins using free ligands is a widely used strategy in molecular biology. Understanding the principles and applications of affinity chromatography is essential for success in CSIR NET, IIT JAM, GATE Biotechnology, and DBT JRF. For expert guidance and structured learning, enroll in Let’s Talk Academy today!