RRC-PRL: Antibody Purification by Affinity Chromatography

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