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

1. Protein Purification strategies

2. Purity requirements – Brief guidelines

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. Three Phase Strategy

6. Proteins Composition

7. Separation Principles in Chromatographic
Purification

8. Gel filtration
Separate by Size

9. Chromatogram of gel filtration
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10. Ion Exchange Chromatography

11. Ion Exchange Type

12. Ion Exchange titration curve
Choice of exchanger group

13. Test tube method to
determine the initial pH

14. Hydrophobic Interaction
Chromatography

15. Different selectivity of HIC media

16. Affinity Chromatography

17. 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

18. Analytical tools
 A rapid and reliable assay for the target
protein
 Purity determination
e.g. SDS-PAGE
 Total protein determination
e.g. colorimetric method

19. Considerations when linking
techniques

20. Logical Combinations of
Techniques

21. Shortcuts – Rapid establishment of
milligram scale purification protocols

22. chromatography computer-controlled
system

23. ÄKTApurifier™ High performance purification
and characterization of proteins

24. ÄKTApurifier™ High performance purification
and characterization of proteins

25. Fraction collectors

26. chromatography computer-controlled system

28. Fraction Collection

29. Chromatogram of cation exchange chromatography
using SP-sepharose Fast Flow column

30. Chromatogram of hydrophobic interaction chromatography
using pheneyl-Sepharose Fast Flow column

31. SDS-PAGE analysis

32. Enzyme purification - SDS-PAGE
analysis

33. Prepacked Column

34. Chromatography Columns

35. Chromatography Media

36. Structure of Separation Media

37. 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

40. ÄKTApurifier™ High performance purification
and characterization of proteins