Antibody affinity purification

Solutions and buffer

PBS
Washing buffer: PBS, 300 mM NaCl
Elution buffer: 50 mM glycine pH 2.5
neutralisation buffer 1 M Na-phosphate pH 7
20% NaN3
  1. prepare a column with 1-10 ml affinity resin (e. g. antigen coupled to Sepharose)
  2. equilibrate the column with PBS (10 volumes)
  3. spin 5-10 ml serum in a ultra centrifuge at 40k for 30 min, 4ºC
  4. apply the cleared serum onto the column.
  5. collect and save the flow through. The binding capacity of the resin may be insufficient. Test the activity of the flow through by western blot. If antibody is still present applied again to the column in a second procedure.
  6. Wash column first with 10 volumes of PBS then with 10 volumes of PBS+300 mM NaCl until the A280 reading does not change anymore.
  7. Prepare eppi cups for collecting the elute fractions with each containing 100 µl (1/10 of fraction size) with neutralising buffer, Note: Some antibodies may precipitate at low pH.
  8. Elute the bound antibody with elution buffer. Collect fractions until the A280 reading drops. Pool all fractions that contain protein.
  9. dialyse the pools against PBS or buffer exchange with NAP10 columns
  10. regenerate column
  11. measure A280 and calculate protein concentration with A280 (1mg/ml IgG) = 1,36.
  12. test the fractions of the antibody in western blots for activity.
  13. if necessary concentrate the antibody with spin columns (Viva spin) with a large cut off size (IgG are big) to at least 1 mg/ml. In case of large volumns, use an ultra-filtration device. Add Na-azid to 0,02% and store at 4ºC

IgG purification

Antibodies can be purified from the serum by affinity chromatography with protein A/G columns. In contrast to affinity purification high-affinity antibodies are not enriched, they are only concentrated. Available columns are the HiTrap-proteinA columns from GE/Amersham.

The procedure is similar as the affinity purification. Antibodies are usually eluted in a single step with low pH buffer.

Regeneration of columns (affinity and protein A

  1. wash with 10 volumes of buffer pH 3,5
  2. wash with 10 volumes of buffer pH 8,5
  3. wash with 10 volumes PBS
  4. wash with PBS + 0,01% NaN3 for storage at 4°C

Options:

The bound antibodies may be eluted in a step-wise manner from the column. As this procedure does not really fractionate the antibodies into low and high affinity antibodies, we stopped doing this.
After washing the column, you may first use a high salt buffer (4 M MgCl2, 50 mM TrisHCl pH 7.5
, then the low pH buffer and finally a high pH buffer (50 mM triethanolamine pH 11.5).
For neutralisation we used a 1 M N-phosphate buffer, which precipitates in the cold room, however.