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Protein_Purification_Techniques_Full_Presentation[1].pptx
- 2. Introduction to ProteinPurification • Process of isolating a specific protein from a complex mixture • Essential for studying protein structure and function • Used in biotechnology, pharmaceuticals, and research • Requires multiple steps for purity and activity retention
- 3. Ultrafiltration • Membrane-based technique •Separates based on molecular size • Concentrates protein samples • Removes salts and small molecules
- 4. Ultrafiltration: Applications • Proteinconcentration prior to chromatography • Buffer exchange • Removal of cell debris • Preparation for further purification steps
- 6. Diafiltration • Variation ofultrafiltration • Involves continuous addition of fresh buffer • Removes unwanted low-molecular-weight compounds • Maintains protein structure by gentle handling
- 7. Diafiltration: Advantages • Efficientbuffer exchange • Scalable for industrial use • Minimizes protein loss • Preserves protein activity
- 9. Chromatographic Techniques Overview • Powerfulmethods for high-resolution purification • Based on different protein properties (size, charge, binding affinity) • Includes SEC, affinity, ion exchange, and more • Focus today: Size Exclusion & Lectin Affinity
- 10. Size Exclusion Chromatography (SEC) •Separates based on molecular size • Also known as gel filtration • Larger proteins elute first • No interaction with stationary phase
- 11. SEC: Applications • Molecularweight estimation • Protein desalting • Final polishing step in purification • High-resolution separation of protein complexes
- 12. Lectin Affinity Chromatography •Selective binding to sugar moieties • Targets glycoproteins • Uses immobilized lectins as ligands • Highly specific and efficient
- 13. Lectin Affinity: Advantages •High specificity for glycosylated proteins • Preserves biological activity • Easy elution with sugar solutions • Ideal for diagnostic and therapeutic proteins
- 14. Comparison of Techniques •Ultrafiltration: Size-based separation for concentration • Diafiltration: Gentle buffer exchange • SEC: High-resolution size separation • Lectin Affinity: Specific for glycoproteins
- 15. Example Workflow • Celllysis and crude extract • Ultrafiltration for concentration • Diafiltration for buffer exchange • SEC and Lectin Affinity for final purification
- 16. Conclusion • Protein purificationis multi-step and method- dependent • Choosing right techniques ensures purity and yield • Ultrafiltration and chromatography are critical tools • Continuous optimization improves results
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