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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!!
AC part 1 Aug 2000 3
Steps of Affinity chromatography
1. Equilibration
MatrixSpecific ligand
Equilibrate the column and the sample to binding conditions.
AC part 1 Aug 2000 4
2. Sample application3. Binding and washing
Target binds
Others wash out
Apply sample under binding conditions.
Wash
AC part 1 Aug 2000 6
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
+
AC part 1 Aug 2000 8
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
+
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 ligandMatrix Affinity tag Target protein
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
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
TipsTips
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
TipsTips
AC part 1 Aug 2000 14
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
AC part 1 Aug 2000 15
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
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 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