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Immunoaffinity Chromatography 55
2.2. Immunoaffinity Chromatography
1. Equilibration buffer: PBS.
2. Elution buffer: 0.1 glycine–HCl, pH 2.25.
3. Cleaning: 100 mM sodium phosphate, 1.5 M NaCl, pH 7.4.
4. Neutralizing buffer: 2 M Tris–HCl, pH 8.6.
5. Fraction collection tubes, 10 ml; screw cap conical centrifuge tubes are convenient.
6. GE Amersham AKTAExplorer or equivalent, including fraction collector.
3. Method
3.1. Coupling
1. Thaw purified antiserum. For a 5-ml pre-packed NHS Sepharose column, the
antiserum should be approximately 5 ml at a concentration of 10 mg/ml. If the
antibody is more dilute, it can be concentrated (see step 2). Affinity-purified
antiserum is typically exchanged (by dialysis or diafiltration) into PBS for storage.
2. If the antiserum is substantially more dilute than 10 mg/ml, use Vivaspin 15R
Hydrosart 30k spin filters to concentrate the antiserum. Add up to 15 ml of
antiserum solution to concentrator and centrifuge at a maximum centrifugal force
of 3000 g. Stop the centrifuge periodically in order to observe the remaining
volume. Do not overconcentrate the antiserum, as this will result in precipitation.
3. Dialyze the antiserum into coupling buffer using dialysis cassettes or dialysis
tubing. Wet the dialysis membrane prior to beginning dialysis. Add the sample
and dialyze while slowly stirring with a magnetic stir bar. A ratio of at least
100:1 should be maintained for the dialysis (1 l of dialysis buffer for each 10 ml
of sample to be dialyzed).
4. Measure total protein concentration using Bradford protein assay. This value will
be used later to calculate coupling efficiency.
5. Wash NHS-activated Sepharose HP column with ice-cold 1 mM HCl. Use 5–10
column volumes, 25–50 ml, at a flow rate of 2 ml/min (60 cm/h). The solution
may be passed using a peristaltic pump, or using a disposable syringe.
6. Inject antiserum solution into column. The antiserum will remain in contact with
the resin for 2–4 h at room temperature or overnight at 2–8ºC. If the entire
antiserum solution is greater than the column volume, recycle the excess through
the column during the immobilization at a flow rate of 2 ml/min (60 cm/h) for
the time period specified above.
7. After immobilization, collect the uncoupled antibody for a Bradford protein assay
to verify coupling efficiency (see Note 4).
8. Remove the uncoupled antibody by passing at least 3 column volumes of blocking
buffer at 2 ml/min (60 cm/h).
9. Replace the blocking buffer with 3 column volumes of coupling wash buffer,
then wash with a further 3 column volumes of blocking buffer. This solution
remains in the column for 30 min at room temperature to block unreacted NHS
sites.