1. CROMATOGRAFIA

5. Anion-exchange Protein at pH higher than pI (by at least 1 unit) is negatively charged and binds to anion-exchange column. Elution is with increasing [Cl - ] (NaCl) or by decreasing pH (harder to control)

6. Cation-exchange Protein at pH lower than pI (by at least 1 unit) is positively charged and binds to cation-exchange column. Elution is with increasing Na + (NaCl) or by increasing pH

7. Setting up ion-exchange 1 test-tube experiments to check pH [salt] for binding [salt] for elution capacity of packing protein’s stability

8. Setting up ion-exchange 2 Set up column and load protein (best to get target protein bound) Wash through unbound protein Apply salt (either in steps or a gradient of increasing conc.)

9. Advantages of ion- exchange 1. large sample volumes OK 2. large amounts of protein OK (1-5 g protein per 100 ml) 3. packings are very robust 4. flexible conditions - huge number of variations

10. Disadvantages of ion-exchange Protein has to be at same pH and [salt] as column. This can be inconvenient for larger volumes dialysis desalting dilution

13. Gel filtration Advantages simple predictable Disadvantages low capacity (volume low) load must be non-viscous Good for 2x to 6x purification

16. Affinity chromatography

17. Affinity tags for purification Recombinant proteins made as constructs to help purification: GST glutathione MBPmaltose biotinavidin polyHisNickel/Zinc

21. Removing the tag Not always necessary (if it doesn’t affect function) Protease site is normally engineered into construct. Make sure that your protein is not a target for the protease! Can use affinity chromatography for digestion to remove the tag.

22. Biotin Avadin factor Xa site factor Xa protease

24. Affinity chromatog - advantages High affinity, with binding in  M, nM or even pM range Binding all or none, so: excellent recovery column dimensions irrelevant Lots of variations possible method, especially if tags used

25. Affinity chromatog- disadvantages Steric hindrance often lowers capacity; spacer arm may overcome. Elution sometimes harsh Specific eluent sometimes needed