Affinity Chromatography

Affinity Chromatography

What is it used for? • Monoclonal and polyclonal antibodies • Fusion proteins • Enzymes • DNA-binding proteins . . . . . . • ANY protein where we have a binding partner!!

Steps of Affinity chromatography 1. Equilibration Equilibrate the column and the sample to binding conditions. Specific ligand Matrix

2. Sample application 3. Binding and washing Apply sample under binding conditions. Wash Target binds Others wash out

4. Desorption and elution Change the eluent to elute the target. Target elutes

+ General elution conditions 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） Reconstituting buffer + Denaturing buffer Usually extremes of pH or chaotropic agents

改变pH至酸性洗脱抗体 LigandSpecificity Protein A Fc region of many IgGs Protein G

+ + Specific eluents Competing ligand in solution Elution of enzymes from Blue Sepharose by free NADH Competing binding substance in solution Elution of glycoproteins from Con A Sepharose by a-D-methylmannoside （伴刀豆球蛋白）（甘露糖苷）

r-Protein Affinity-tagged fusion proteins Specific ligand Affinity tag Target protein Matrix • 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 Cleavage site

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

Sample preparation Tips • 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

Sample application Tips • 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

Affinity chromatography is easy Simple to do Concentrates High purity

Purification of monoclonal mouse IgG1 Sample: Cell culture supernatant containing mouse IgG1 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 SDS-PAGE MWr 94000 67000 43000 30000 20100 14400 1: LMW 2: Starting material, diluted 10 X 3: Eluted IgG1 pool, diluted 5 X

Purification of recombinant His-tagged protein Sample: Clarified homogenate of E. coli expressing His fusion protein Column: HiTrap Chelating 1 ml charged with Ni2+ 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 SDS-PAGE MWr 94000 67000 43000 30000 20100 14400 1: LMW 2: Starting material, diluted 20 X 3: Eluted peak 1, diluted 20 X 4: Eluted peak 2, diluted 20 X

KD 94 67 43 30 20.1 14.4 Purification of recombinant GST-tagged protein 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

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

Affinity Chromatography