Ammonium Sulfate Precipitation Protocol
- Allow serum or ascitic fluid to thaw, determine total volume, and centrifuge at 3000g for 30 minutes.
- Transfer sample to beaker containing a stir bar and place on magnetic stirrer.
- While sample is stirring, slowly add saturated ammonium sulfate to bring final concentration to 50% saturation.
- Volume of ammonium sulfate needed is equal to volume of sample.
- Adding the ammonium sulfate very slowly ensures that local concentration around the site of addition does not exceed the desired salt concentration.
- Once total volume of ammonium sulfate is added, move beaker to 4°C for 6 hours or overnight.
- Transfer to conical tube and centrifuge the precipitate at 3000g for 30 minutes.
- 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.
- 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.
- Remove antibody solution from the tubing and centrifuge to remove any remaining debris.
- Determine the concentration and store at -80°C for long term storage.