Affinity Purification Protocol:
Please note that this protocol is designed for affinity purifying additional serum using the column that we have created for you:

  1. Dilute 10ml of clear filtered antiserum 1:1 with PBS pH7.2, apply to the affinity column at approximate rate of 0.3ml/min and monitor absorbance of flow thru at 280nm.
  2. Collect unbound material / flow thru. Do not discard flow-thru until ELISA results show that specific antibody has been removed. Wash with PBS until A280 reaches baseline.
  3. Elute bound antibodies with 0.2M glycine, pH1.85 and collect eluate until absorbance returns to baseline.
  4. Neutralize all collected fractions with 1M Tris-HCl, pH8.5 immediately after collection. The fractions can be collected into tubes containing the Tris. The time the eluted antibodies are at low pH should be as short as possible.
  5. Determine the OD at 280nm by spectrophotometer and determine the total OD recovered.
  6. Conduct ELISA analysis with the corresponding antigen to confirm the recovered antibody and the removal of all the antibodies from the original serum.
  7. Concentrate the antibody to ~1-2mg/ml and dialyze against PBS with 0.02% sodium azide. Purified antibody can be stored refrigerated or aliquoted and frozen. Avoid repeated freeze-thaw cycles.
  8. The column should be stored refrigerated in PBS, 0.02% sodium azide. Do not freeze.

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