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Ammonium Sulfate Fractionation of Antibodies 1. Start with 5-10 mls of ascites fluid. Spin 30 minutes at 5000 rpm in HB-4 at 2C.
 

Summary: 100
Ammonium Sulfate Fractionation of Antibodies
1. Start with 5-10 mls of ascites fluid. Spin 30 minutes at 5000 rpm in HB-4 at 2C.
2. Transfer supernatant to small beaker.
3. Slowly (over 1-2 minutes), with continuous stirring, add 1/3 volume of saturated ammonium
sulfate to bring final concentration to 25%.
4. Cover beaker with Parafilm and place on ice for 2-4 hours. This step can go overnight at 4C.
5. Spin 30 minutes at 5000 rpm in HB-4 at 2C.
6. Transfer supernatant to small beaker.
7. Slowly (over 1-2 minutes), with continuous stirring, add 1/3 volume of saturated ammonium
sulfate to bring final concentration to 50%.
8. Cover beaker with Parafilm and place on ice for 2-4 hours. This step can go overnight at 4C.
9. Spin 30 minutes at 5000 rpm (~4000 g) in HB-4 at 2C.
10. Carefully remove and save all of the supernatant. The antibody is in the pellet fraction.
11. Resuspend pellet in 1/2 of the original volume (2.5-5 mls) of PBS. Avoid bubbles and foaming.
12. Transfer to dialysis tubing (SpectraPor 2, 12-14 kDal molecular weight cut off). Use twice as
much dialysis tubing as needed for the antibody in PBS. This will allow enough space for expansion
during dialysis. Carefully place tubing clips at the ends.
13. Dialyze overnight at 4C in ~100 volumes (~500 mls) of PBS. Change buffer three times. Allow
at least a few hours between changes.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine