By Amersham Biosciences
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Additional resources for Antibody Purification
Prepacked 1 ml or 5 ml columns. rProtein A Sepharose 4 Fast Flow* Human IgG > 50 mg Mouse IgG 8–20 mg Enhanced binding capacity. Supplied as a suspension ready for column packing. Protein A Sepharose 6MB Human IgG > 5 mg For purification of cells coated with antibodies. MabSelect (recombinant protein A ligand) Human IgG, approx. 30 mg Purification of IgG, IgG fragments and sub-classes. Fast processing of large sample volumes. Retains high binding capacity at high flow rates. Supplied as a suspension ready for column packing.
Keep on ice. Pre-clearing (optional) 1) Add 50–100 µl of prepared Protein A Sepharose 4 Fast Flow or Protein G Sepharose 4 Fast Flow suspension (50% slurry) to 1 ml cell lysate in an Eppendorf tube. 2) Gently mix for 1 hour at +4 °C. 3) Centrifuge at 12 000 x g for 20 seconds. Save the supernatant. Couple antigen to antibody 1) Aliquot samples (500 µl) into new Eppendorf tubes. 1–1 µl) or purified monoclonal or polyclonal antibodies (add the volume corresponding to 1–5 µg). For controls, use non-immune antibodies that are as close to the specific antibody as possible (for example, polyclonal serum should be compared to normal serum from the same species).
Ligand coupling 1. Wash out the isopropanol with 3 x 2 column volumes of ice-cold 1 mM HCl. 2. Inject one column volume of ligand solution onto the column. 3. Seal the column and leave for 15–30 minutes at +25 °C (or 4 hours at +4 °C). Do not use excessive flow rates (maximum recommended flow rates are 1 ml/min, equivalent to approximately 30 drops/min when using a syringe, with HiTrap 1 ml and 5 ml/min, equivalent to approximately 120 drops/min when using a syringe, with HiTrap 5 ml). The column contents can be irreversibly compressed.
Antibody Purification by Amersham Biosciences