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54 Gallant et al.
monoclonal antibodies or purified polyclonal antisera maybe used (2). Because
elution from a polyclonal column often requires extremely low pH, some
consideration should be given to the stability of the protein of interest below
pH 3 if that strategy is pursued. In contrast to the use of a polyclonal column,
use of a monoclonal column usually allows for milder elution conditions; this
is achieved by screening for a monoclonal antibody of intermediate affinity.
A number of companies (see Note 1) offer economical production of
monoclonal antibodies or purified polyclonal antisera (3). These antibody
sources can be purified using standard protocols with Protein A and Protein G
affinity chromatography (2). Subsequently, the antibodies may be covalently
attached to activated resin using a range of chemistries (4). Two of the most
common approaches are attachment through surface lysines and site-directed
attachment through the carbohydrate chains (see Note 2).
In the following method, a purified polyclonal antiserum is coupled to
Amersham Biosciences NHS-activated Sepharose 4 HP (5,6). The resin is a
34-μm average particle size agarose resin appropriate for protein purification
using low pressure chromatography equipment. The resulting immunoaffinity
resin is used to purify a recombinant protein from mammalian cell culture.
2. Materials
2.1. Coupling
1. NHS-activated Sepharose HP column, 5 ml (Amersham/GE Healthcare P/N 17-
0717-01) (see Note 3).
2. Protein G purified antiserum (2). A concentration of >10 mg/ml is convenient;
lower concentrations may be used with recirculation during immobilization.
3. Vivascience Vivaspin 15R Hydrosart 30k spin filters (VS15RH21) or equivalent.
4. Phosphate-buffered saline (PBS).
5. Slide-A-Lyzer Dialysis Cassettes (Pierce, http://www.piercenet.com) or equivalent
dialysis tubing. The 3–12 ml size will be convenient for 5 ml of antiserum.
6. Pierce Coomassie Plus protein assay kit (23236) or equivalent.
7. Solution of hydrochloric acid, 1 mM, on ice.
8. Disposable syringes, 10 ml and 60 ml. Alternatively, a peristaltic pump may
be used to pass solutions over the column. The use of syringes can make the
immobilization more convenient; however, a flow rate of 2 ml/min should not
be exceeded in order to insure that the resin is not damaged by high pressure.
9. Coupling buffer: 0.2 M ammonium bicarbonate, 0.5 M NaCl, pH 8.3.
10. Blocking buffer: 0.2 M Tris–HCl, 0.5 M NaCl, pH 8.3 (we have also used 0.2
M glycine, 0.5 M NaCl, pH 8.3 for this step).
11. Coupling wash buffer: 0.1 M sodium acetate, 0.5 M NaCl, pH 4.
12. Storage buffer: PBS with 0.05% sodium azide or 0.2 M imidazole, 0.5 M
NaCl, pH 7.