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Affinity Chromatography

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What is it used for?

• Monoclonal and polyclonal antibodies

• Fusion proteins

• Enzymes

• DNA-binding proteins . . . . . .

• ANY protein where we have a binding partner!!

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Steps of Affinity chromatography

1. Equilibration

MatrixSpecific ligand

Equilibrate the column and the sample to binding conditions.

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2. Sample application3. Binding and washing

Target binds

Others wash out

Apply sample under binding conditions.

Wash

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4. Desorption and elution

Target elutes

Change the eluent to elute the target.

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Reconstituting

buffer

+

Changing buffer conditionsUsually 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 bufferUsually extremes of pH or chaotropic agents

General elution conditions

+

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Ligand Specificity

Protein A Fc region of many IgGs

Protein G

改变 pH至酸性洗脱抗体

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Competing binding substance in solutionElution of glycoproteins from Con A Sepharose by Dmethylmannoside （伴刀豆球蛋白） （甘露糖苷）

Specific eluents

+

Competing ligand in solutionElution of enzymes from Blue Sepharose by free NADH

+

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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 ligandMatrix Affinity tag Target protein

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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

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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

TipsTips

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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

TipsTips

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Affinity chromatography is easy

Simple to do

Concentrates

High purity

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Sample: Cell culture supernatant containing mouse IgG1

Column: HiTrap Protein G 1 mlBinding buffer: 20 mM sodium phosphate, pH 7.0Elution buffer: 0.1 M glycine-HCl, pH 2.7System: ÄKTAprime, 1.0 ml/min

94000

67000

14400

20100

30000

43000

MWr

1: LMW2: Starting material, diluted 10 X3: Eluted IgG1 pool, diluted 5 X

SDS-PAGE

Purification of monoclonal mouse IgG1

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Sample: Clarified homogenate of E. coli expressing His fusion proteinColumn: HiTrap Chelating 1 ml charged with Ni2+

Binding buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4Elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4System: ÄKTAprime, 1 ml/min

94000

67000

14400

20100

30000

43000

MWr

1: LMW2: Starting material, diluted 20 X3: Eluted peak 1, diluted 20 X4: Eluted peak 2, diluted 20 X

SDS-PAGE

Purification of recombinant His-tagged protein

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94

67

43

30

20.1

14.4

KD

Sample: 8 ml cytoplasmic extract from E. coli expressing a GST fusion proteinColumn: GSTrap 1 mlBinding buffer: PBS, pH 7.3Elution buffer: 50 mM Tris-HCl, pH 8.0 with 10 mM reduced glutathioneSystem: ÄKTAexplorer 10, 1 ml/min

Lane 1: Low Molecular Weight (LMW) Calibrationkit, reducedLane 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

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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