Description

Ammonium sulfate precipitation is one of the most commonly used methods for protein purification from a solution. In solution, proteins form hydrogen bonds with water molecules through their exposed polar and ionic groups. When high concentrations of small, highly charged ions such as ammonium sulfate are added, these groups compete with the proteins to bind to the water molecules. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation. Critical factors that affect the concentration at which a particular protein will precipitate include: the number and position of polar groups, molecular weight of the protein, pH of the solution, and temperature at which the precipitation is performed. The concentration at which antibodies precipitate varies among species; most rabbit antibodies precipitate with a 40% saturated solution, whereas mouse antibodies require 45-50% saturation.

Protocol

  1. Allow serum or ascitic fluid to thaw, determine total volume, and centrifuge at 3000g for 30 minutes.
  2. Transfer sample to a beaker containing a stir bar and place on a magnetic stirrer.
  3. While the sample is stirring, slowly add saturated ammonium sulfate to bring the final concentration to 50% saturation.
    1. Volume of ammonium sulfate needed is equal to the volume of sample.
    2. Adding the ammonium sulfate very slowly ensures that local concentration around the site of addition does not exceed the desired salt concentration.
  4. Once the total volume of ammonium sulfate is added, move beaker to 4°C for 6 hours or overnight.
  5. Transfer to conical tube and centrifuge the precipitate at 3000g for 30 minutes.
  6. Carefully remove and discard supernatant. Invert conical tube and drain well. For serum or ascites, resuspend pellet in 30%-50% of the starting volume in 1XPBS. For monoclonal antibody tissue culture supernatants, resuspend pellet in 10% of the starting volume in 1X PBS.
  7. Transfer antibody solution to dialysis tubing and dialyze versus three changes of 1XPBS/0.08% Sodium Azide. Be sure to allow enough space for expansion of the antibody solution during dialysis. Normally twice the re-suspended volume is sufficient.
  8. Remove antibody solution from the tubing and centrifuge to remove any remaining debris.
  9. Determine the concentration and store at -80°C for long-term storage.