Affinity Purification Protocol:
Please note that this protocol is designed for affinity purifying additional serum using the column that we have created for you:
- 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.
- 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.
- Elute bound antibodies with 0.2M glycine, pH1.85 and collect eluate until absorbance returns to baseline.
- 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.
- Determine the OD at 280nm by spectrophotometer and determine the total OD recovered.
- Conduct ELISA analysis with the corresponding antigen to confirm the recovered antibody and the removal of all the antibodies from the original serum.
- 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.
- The column should be stored refrigerated in PBS, 0.02% sodium azide. Do not freeze.
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