- Ammonium sulfate precipitation outline
- Ammonium sulfate precipitation methods
- Ammonium sulfate saturation methods
Ammonium Sulfate Precipitation Overview
Ammonium sulfate precipitation is a non-specific purification method that involves simple purification of proteins from a solution. Even though polar and ionic groups of proteins form hydrogen bonds with water in solution, when adding small ions such as ammonium sulfate, water molecules are removed from proteins, allowing protein to precipitate.
Ammonium Sulfate Precipitation Method
- (1) Centrifuge ascites fluid at 4℃ for 30 minutes at 3,000rpm and transfer the supernatant into a flask.
- (2) Stir the flask containing the antibody (4℃ on ice) and slowly add 1/3 volume of saturated
Ammonium sulfate (final concentration 25%) and stir for 2-4 hours.
- (3) Transfer to a high speed centrifuge tube and centrifuge at 4℃ for 30 minutes at 8,000rpm (Appx. 4,000G).
- (4) Stir the flask containing the antibody (4℃ on ice) and slowly add 1/3 volume of saturated
Ammonium sulfate (final concentration 50%) and stir for 2-4 hours.
- (5) Transfer to a high speed centrifuge tube and centrifuge at 4℃ for 30 minutes at 8,000rpm (Appx. 4,000G).
- (6) Store the supernatant and dry the pellet.
- (7) Dissolve pellet by adding 1XPBS (50% of original volume).
- (8) Transfer to a dialysis tube and stir for 30 minutes at 4℃ in 1XPBS. (Appx. 100 volumes)
- (9) Replace the PBS and stir for 1 hour at 4℃.
- (10) Replace with 2XPBS (Appx. 100 volumes) and stir overnight at 4℃.
(When conducting DEAE chromatography purification, use 10mM Tris-HCl, pH8.5 buffer.)
- (11) Add thimerosal to 0.03% and freeze at -70℃.
Ammonium Sulfate Saturation Method
- (1) Dissolve 80g ammonium sulfate in 100ml ddH2O. (Appx. 4.1M)
- (2) Dissolve by heating slightly.
- (3) Cool at room temperature.
- (4) Adjust to pH7.0 by adding NH4OH or H2SO4.
- (5) Store at 4℃ following filter sterilization.