Protein Purification Techniques Lecture Notes

Introduction to Protein Purification

• Objective: Isolate a single type of protein (target protein) from a biological tissue or culture, free of contaminants and isoforms.
• Applications:
  • Biopharmaceuticals
  • Food supplements
  • Detergent industry
  • Scientific research (characteristics, structures, and functions of proteins)

Preliminary Considerations

• Understand the target protein’s properties (chemical, structural, functional).
• Identify unique features (size, shape, charge, pI, solubility, etc.) to differentiate it from others.

Initial Steps

• Destruction of cells using chemical (osmotic shock) or mechanical (ultrasonification) methods.
• Centrifugation to separate cell debris.
• Ensure protein solubility and stability using appropriate buffers.

Protein Purification Techniques

Precipitation Methods

• Objective: Separate proteins based on solubility.
• Salting Out:
  • Uses salt (e.g., aluminum sulfate) to decrease protein solubility.
  • Environmental friendly and low-cost; often combined with other methods for enhanced purification.

Chromatography

• General Principle: Proteins separated based on interactions with stationary and mobile phases.

Affinity Chromatography

• Basis: Affinity of proteins to ligands or affinity tags.
• Example: His-tag with Ni2+ coordination.
• Benefit: Highly specific and effective.

Ion Exchange Chromatography

• Basis: Protein charge at certain pH.
• Types: Anion exchange (negative proteins) and cation exchange (positive proteins).

Hydrophobic Interaction Chromatography

• Basis: Exploits hydrophobic regions in proteins.
• Process: Use salt to enhance hydrophobic interactions, then reduce to elute.

Size Exclusion Chromatography

• Basis: Separation based on protein size.
• Process: Larger proteins elute faster; smaller proteins elute slower due to pore interaction.

Filtration Methods

• Dialysis:
  • Uses semi-permeable membrane to reduce ions or change pH.
  • Does not separate proteins but adjusts solution conditions.

Electrophoresis

• Process: Separates proteins by charge, size, and shape.
• Example: Native PAGE (polyacrylamide gel electrophoresis).
• Characteristics:
  • Separates by charge density, molecular weight, and shape.

Validation Methods

• Techniques:
  • SDS-PAGE
  • Western blot
  • Spectrophotometric assays

Conclusion

• Protein purification involves multiple techniques, often combined for efficacy.
• Method choice depends on desired purity and application.

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• Additional Resources: Check out more videos linked in the presentation for practical applications of techniques like affinity chromatography.
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