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> Summary of Rabbit Immunization Procedures

    > Affinity Purifying Antibody from Rabbit Serum
    > Purifying Antibodies Made using a GST-fusion Protein


Affinity Purifying Antibody from Rabbit Serum

If your antigen was a fusion protein then you will usually need to purify your antibody in two steps.  The first step is to run the serum over a column containing the fusion partner (usually GST in our lab but many other fusion partners are used).  This step removes the antibodies that recognize the fusion partner.  Obviously if your fusion partner was Protein A or Protein G you cannot do this step as your specific antibody will also bind to Protein A.  You can use Protein L as rabbit antibodies bind poorly to Protein L.
The second column contains beads coupled to the fusion protein that you used as antigen.  The antibodies you want flow-through the first column and stick to the second column.  Because they are so similar, the instructions here are for the second column.

  1. Wash Sepharose beads that have antigen coupled to them as follows:
    1. 3X PBS with 1mM PMSF and 1X PIN
    2. 2X with 0.2M glycine pH 2.65 (to get rid of stuff on the columns)
    3. 2X with 0.1M NaHCO3 / 0.5 NaCl pH 8.3 + PIN + PMSF
    4. 2X with PBS + PMSF +PIN
  2. Check pH and adjust to approx. 7.5 if necessary
  3. Incubate 0.5 ml beads with 3ml serum + 4ml PBS, overnight at 4°C.
  4. Pour beads into column and let settle.
  5. Collect the flow through (will contain unbound proteins from serum).
  6. Wash twice with 10mL of PBS.
  7. Elute bound antibodies with 2mL of 0.2M glycine pH 2.6.  Collect 200uL per fraction into 1.5mL tubes containing 60uL of 1M Tris pH 8.0.  This neutralizes the glycine buffer.
  8. Do a Bradford assay to check for protein peaks.
  9. Run a gel for the peak fractions to make sure that they contain only antibody and then combine these fractions. Test antibody.  It is often a good idea to dialyze the buffer down to 50 mM Tris HCl pH 8.0, add 50% glycerol and store at -20°C. 
  10. Test antibody!