Download PDF by Amersham Biosciences: Antibody Purification

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4. Centrifuge at 10 000 x g, at +4 °C for 25 minutes to precipitate the lipids. 5. Collect the supernatant containing the IgY. 6. 5 M. 7. 5. 8. 45 µm filter immediately before applying it to the column. Purification 1. Wash the column with at least 5 column volumes of binding, elution and wash buffer. 2. Equilibrate with 5 column volumes of binding buffer. 3. Apply the sample. 4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent, as monitored at A280.

45 µm filters is recommended. Optimal binding and elution conditions for purification of the target protein must be determined separately for each ligand (see below for elution buffer suggestions). Literature references and textbooks may give good guidelines. The general protocol given here can be used as a guideline for a preliminary separation. 48 For the first run, perform a blank run to ensure that any loosely bound ligand is removed. 45 µm filter. If the sample is too viscous, dilute with binding buffer.

E. BSA, gelatine, acetone powders). Spin the lysate at 100 000 x g for 30 minutes to remove aggregated proteins before addition of the antibody. Spin the antibody at 100 000 x g for 30 minutes to remove particulate matter. Spin the antigen-antibody complex at 10 000 x g for 10 minutes prior to addition of medium to remove protein aggregates. Try a different antibody. 2% SDS or 1% Tween 20. Alternate between high and low salt wash buffer, or wash the beads with distilled water. Prolong washing times and/or increase the number of washes.

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Antibody Purification by Amersham Biosciences


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