Antibody purification

Dr E. Buxbaum EB15 at le.ac.uk
Mon Mar 3 06:00:28 EST 1997


GMEACHAM at bmg.bhs.uab.edu wrote:
>Does anyone have a protocol or reference for precipitating Antisera with 
>Ammonium Sulfate? Info like optimal concentrations of AmSo4, best buffer 
>to dialyze in, etc. would be useful. Thanks in advance.
>

Precipitation is usually carried out with 45% saturation of ammonium 
sulfate (i.e. a concentrated solution is called 100%). 45% saturation 
is equivalent to 277 g/l. Add slowly in small portions to your sample, 
while constantly stirring on ice. Leave stirring for 30 min, then spin, 
keeping the sample cold all the time. 

The buffer used for dissolving and dialysing the pellet obviously depends 
on what you need the antibody for. Phosphate (PBS) or Tris (TBS) buffered 
saline are probably most commonly used. If however you want to further 
purify by ion exchange chromatography, you need a low ionic strength 
buffer (10 mM Tris-HCl pH 7 or similar). If instead you want to use 
hydrophobic interaction chromatography, you need a high salt buffer 
instead (add as much ammonium sulfate as possible without precipitating 
the antibody).




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