1. AC part 1 Aug 2000 1 Affinity Chromatography

2. AC part 1 Aug 2000 2 What is it used for? Monoclonal and polyclonal antibodies Fusion proteins Enzymes DNA-binding proteins...... ANY protein where we have a binding partner!!

3. AC part 1 Aug 2000 3 Steps of Affinity chromatography 1. Equilibration Matrix Specific ligand Equilibrate the column and the sample to binding conditions.

4. AC part 1 Aug 2000 4 2. Sample application 3. Binding and washing Target binds Others wash out Apply sample under binding conditions. Wash

5. AC part 1 Aug 2000 5 4. Desorption and elution Target elutes Change the eluent to elute the target.

6. AC part 1 Aug 2000 6 Reconstituting buffer + Changing buffer conditions Usually decrease pH, increase ionic strength or decrease polarity adding up to 10 % dioxane （二氧六环 ） or up to 50 % ethylene glycol （乙二 醇 ） Denaturing buffer Usually extremes of pH or chaotropic agents General elution conditions +

7. AC part 1 Aug 2000 7 LigandSpecificity Protein AFc region of many IgGs Protein G 改变 pH 至酸性洗脱抗体

8. AC part 1 Aug 2000 8 Competing binding substance in solution Elution of glycoproteins from Con A Sepharose by  D  methylmannoside （伴刀豆球蛋白） （甘露糖苷） Specific eluents + Competing ligand in solution Elution of enzymes from Blue Sepharose by free NADH +

9. AC part 1 Aug 2000 9 Affinity-tagged fusion proteins The affinity-tag binds to a specific ligand 'Only' the tagged fusion protein binds to the ligand Binding usually possible in 8 M Urea or 6 M GuHCl (depends on tag ， eg. His-Tag) A protease cleavage site allows the tag to be removed after purification Purity typically >90% in one step r-Protein Cleavage site Specific ligand Matrix Affinity tagTarget protein

10. AC part 1 Aug 2000 10 The main stages in affinity chromatography Prepare a gel to bind the target specifically Equilibrate gel and sample to binding conditions Apply the sample and wash out contaminants Desorb and elute the target Re-equilibrate the gel to binding conditions

11. AC part 1 Aug 2000 11 Sample preparation Filter or centrifuge to remove particles Use a desalting column to adjust pH, buffer salts and additives to promote binding Make sure that components known to interfere with binding are absent Tips

12. AC part 1 Aug 2000 12 Sample application Follow the standard protocol Wash the column before applying sample Binding buffer: usually neutral pH Establishment of equilibrium: – Strong affinity and fast equilibrium: High flow rate – Weak affinity and/or slow equilibrium: Low flow rate Tips

13. AC part 1 Aug 2000 13 Affinity chromatography is easy Simple to do Concentrates High purity

14. AC part 1 Aug 2000 14 Sample: Cell culture supernatant containing mouse IgG 1 Column: HiTrap Protein G 1 ml Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1 M glycine-HCl, pH 2.7 System:ÄKTAprime, 1.0 ml/min 94000 67000 14400 20100 30000 43000 MWr 1: LMW 2: Starting material, diluted 10 X 3: Eluted IgG 1 pool, diluted 5 X SDS-PAGE Purification of monoclonal mouse IgG 1

15. AC part 1 Aug 2000 15 Sample: Clarified homogenate of E. coli expressing His fusion protein Column: HiTrap Chelating 1 ml charged with Ni 2+ Binding buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4 Elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4 System:ÄKTAprime, 1 ml/min 94000 67000 14400 20100 30000 43000 MWr 1: LMW 2: Starting material, diluted 20 X 3: Eluted peak 1, diluted 20 X 4: Eluted peak 2, diluted 20 X SDS-PAGE Purification of recombinant His-tagged protein

16. AC part 1 Aug 2000 16 94 67 43 30 20.1 14.4 KD Sample: 8 ml cytoplasmic extract from E. coli expressing a GST fusion protein Column: GSTrap 1 ml Binding buffer: PBS, pH 7.3 Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathione System: ÄKTAexplorer 10, 1 ml/min Lane 1: Low Molecular Weight (LMW) Calibration kit, reduced Lane 2: Cytoplasmic extract of E.coli expressing GST fusion protein Lane 3: GST fusion protein eluted from GSTrap 1 ml Purification of recombinant GST-tagged protein

17. AC part 1 Aug 2000 17 Summary Affinity chromatography is simple to do & can give high purity in one step Group-specific ligands are the easiest to use General elution conditions are often harsh Specific eluents are kinder