Affinity Chromatography - Department of Molecular and Cellular ...

Purification examples
Figure 11 shows the purification of mouse monoclonal IgG 1 on HiTrap Protein G HP 1 ml.
The monoclonal antibody was purified from a hybridoma cell culture supernatant.
Sample: 12 ml mouse IgG 1 hybridoma cell culture supernatant
Column: HiTrap Protein G HP, 1 ml
Flow:
1.0 ml/min
Binding buffer: 20 mM sodium phosphate, pH 7.0
Elution buffer: 0.1 M glycine-HCI, pH 2.7
Electrophoresis: SDS-PAGE, PhastSystem, PhastGel
Gradient 10–15, 1 µl sample, silver stained
Immunodiffusion: 1% Agarose A in 0.75 M Tris,
0.25 M 5,5-diethylbarbituric acid,
5 mM Ca-lactate, 0.02% sodium azide, pH 8.6
Immunodiffusion
A 280 nm
Binding
buffer
Elution Binding
buffer buffer
5.0
SDS PAGE
2.5
0
pool I
pool II
5 10 15 20 25 30 ml
M r
97 000
66 000
45 000
30 000
20 100
14 000
Lane 1 2 3 4
Lane 1. Low Molecular Weight
Calibration Kit, reduced
Lane 2. Mouse hybridoma cell
culture fluid, non-reduced,
diluted 1:10
Lane 3. Pool I, unbound material,
non-reduced, diluted 1:10
Lane 4. Pool II, purified mouse IgG 1 ,
non-reduced, diluted 1:10
Fig. 11. Purification of monoclonal mouse IgG 1
on HiTrap Protein G HP, 1 ml.
Figure 12 shows the purification of recombinant mouse Fab fragments, expressed in
E. coli, using Protein G Sepharose 4 Fast Flow. Chimeric, non-immunogenic "humanized"
mouse Fab, Fab' and F(ab') 2 fragments are of great interest in tumour therapy since they
penetrate tumours more rapidly and are also cleared from the circulation more rapidly than
full size antibodies.
A 280 nm
3.5
2.5
1.5
0.5
0
0.0
UV 280 nm
Conductivity
pH
Elution
buffer
10.0 20.0 30.0 40.0 ml
Sample: Recombinant Fab fragment
from E. coli.
Medium: Protein G Sepharose 4
Fast Flow (1 ml)
Flow: 0.2 ml/min (60 cm/h),
or 0.3 ml/min (90 cm/h)
Binding buffer: 0.15 M NaCl,
10 mM sodium phosphate,
10 mM EDTA, pH 7.0
Elution buffer: 0.5 M ammonium acetate,
pH 3.0
Wash buffer: 1 M acetic acid, pH 2.5
Fig. 12. Purification of recombinant Fab fragments directed to the envelope protein gp120 of HIV-1 (anti-gp120 Fab),
expressed in E. coli.
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