Protocol: Antibody Purification by Affinity Chromatography

1. Preparing the gel
   • 1gm freeze dried Sepharose 4 B in 50ml 1mM HCl buffer, mix.
   • Spin at low speed, remove supernatant.
   • Wash 15 min with 1mM HCl , spin, discard supernatant --- Repeat twice.
2. Coupling the ligand
   • Dissolve 10mg synthetic peptide(ligand) in 5ml coupling buffer ( 0.1M NaHCO3 pH 8.3, containing 0.5ml NaCl ).
   • Mix the coupling buffer containing the ligand with the swollen gel . Stir gently for 1 hr.
   • Spin at low speed, discard supernatant.
   • Wash excess ligand with 20ml coupling buffer.
   • Block remaining active groups by transfering gel to 0.1M Tris HCl buffer pH 8.0, stand for 2 hrs.
   • Wash the gel with 0.1M acetate buffer pH 4.0 containing 0.5M NaCl, spin, discard supernatant.
   • Wash the gel with 0.1M Tris HCl buffer pH 8.0 containing 0.5M NaCl, spin, discard supernatant.
   • Transfer the gel into PBS.
   • Pack the column by pouring the gel into a vertically held column.
   • Wash the column with 100 bed volumes of PBS.
3. Binding
   • Filter 15ml rabbit antiserum through 0.2um filter.
   • Dilute antiserum with PBS to 50ml.
   • Load the antiserum to the affinity column.
   • Wash with 20ml PBS.
   • Wash with 20ml Tris buffer, pH 8.0 (50mM Tris-Cl, pH 8.0; 0.1% Triton X-100; 0.5 M NaCl).
   • Wash with 20ml Tris buffer, pH 9.0 (50mM Tris-Cl, pH 9.0; 0.1% Triton X-100; 0.5 M NaCl).
   • Wash with 20ml Sodium phosphate buffer ( 50mM Sod. Phosphate, pH 6.3; 0.1% Triton X-100; 0.5 M NaCl ).
4. Elution
   • Elute antibodies with 20ml glycine buffer, pH 2.5 (50mM glycine-HCl, pH 2.5; 0.1% Triton X-100; 0.15 M NaCl ).
   • Collect fractions into tubes containing 4ml 1M Tris-Cl, pH 9.0.
   • Wash column with 20ml PBS.
5. Desalting
   • Use PD-10 column to desalt the antibodies, using PBS as the desalt buffer. Lyophilize the Antibody.