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Protein purification stratigies

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training course in department of biology .Faculty of science.University of Kufa

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Protein purification stratigies

  1. 1. Protein Purification strategies
  2. 2. Purity requirements – Brief guidelines
  3. 3. o Preparation for protein isolation o Protein extraction and solubilization o Protein concentration determination o Concentrating protein solution o Ammonium sulfate precipitation o ultrafiltration, Freeze-drying (Lyophilization) , dialysis o Column chromatography for purification of protein Steps of protein purification
  4. 4. Three Phase Strategy
  5. 5. Proteins Composition
  6. 6. Separation Principles in Chromatographic Purification
  7. 7. Gel filtration Separate by Size
  8. 8. Chromatogram of gel filtration •
  9. 9. Ion Exchange Chromatography
  10. 10. Ion Exchange Type
  11. 11. Ion Exchange titration curve Choice of exchanger group
  12. 12. Test tube method to determine the initial pH
  13. 13. Hydrophobic Interaction Chromatography
  14. 14. Different selectivity of HIC media
  15. 15. Affinity Chromatography
  16. 16. Summary of affinity chromatography  Affinity chromatography is simple to do and can give high purity in one step  Group-specific ligands are the easiest to use  Design the elution scheme  General elution conditions are often harsh  Specific eluents are kinder
  17. 17. Analytical tools  A rapid and reliable assay for the target protein  Purity determination e.g. SDS-PAGE  Total protein determination e.g. colorimetric method
  18. 18. Considerations when linking techniques
  19. 19. Logical Combinations of Techniques
  20. 20. Shortcuts – Rapid establishment of milligram scale purification protocols
  21. 21. chromatography computer-controlled system
  22. 22. ÄKTApurifier™ High performance purification and characterization of proteins
  23. 23. ÄKTApurifier™ High performance purification and characterization of proteins
  24. 24. Fraction collectors
  25. 25. chromatography computer-controlled system
  26. 26. Fraction Collection
  27. 27. Chromatogram of cation exchange chromatography using SP-sepharose Fast Flow column
  28. 28. Chromatogram of hydrophobic interaction chromatography using pheneyl-Sepharose Fast Flow column
  29. 29. SDS-PAGE analysis
  30. 30. Enzyme purification - SDS-PAGE analysis
  31. 31. Prepacked Column
  32. 32. Chromatography Columns
  33. 33. Chromatography Media
  34. 34. Structure of Separation Media
  35. 35. Primary sequence information and amino acid composition from blots (Blotting to PVDF) 1. Blotting is a technique for the electrophoretic transfer of DNA, RNA or protein to a suitable membrane. 2. For protein sequencing and amino acid analysis, the proteins are transferred to polyvinylidene difluoride (PVDF) membrane. 3. Bands visualized on the blot with Coomassie Brilliant Blue R-250 are excised, cut into smaller pieces, washed extensively with deionized water and used directly for N-terminal sequencing or amino acid analysis
  36. 36. ÄKTApurifier™ High performance purification and characterization of proteins

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