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Star Republic: Guide for Biologists

Purification --- affinity (I)

Edited by Chang Zhu
1. Coupling of antigen to a solid support:

  • Coupling of GST-protein to glutathione beads
  • Coupling of peptides to Sepharose beads

    2. Wash the beads with PBS or PBS/0.01% Tween X-100.

    3. Apply antibody solutions.

    4. Wash with PBS or PBS/0.01% Tween X-100.

    5. If binding in batch, transfer to a column.

    6. Elute with 100 mM glycine, pH 2.5-2.8. Collecting fractions. Immediately neutralize with 1/10 faction volume of 1 M Tris-HCl pH 8.0 in the collection tubes.

    7. Read A280 and pool antibody containing fractions.

    8. Dialyze against PBS/0.02% sodium azide.

    9. The affinity column can be regenerated with extensive wash of 0.2 M glycine (pH 2.8), followed by extensive wash of a pH 7.0-8 buffer.