The saturation of ammonium sulfate precipitation is 50%, then ran SDS PAGE followed by Coomassie blue staining, i got nonspecific bands at different sizes along with my protein of interest. Can anyone suggest how to get rid from those nonspecific bands (note: My protein don't have any tag)
Ammonium sulphate precipitation is great as an initial purification step, but, as with your example, other proteins will frequently co-purify. You'll need to use a subsequent purification step such as ion exchange, hydrophobic interaction or size exclusion chromatography to remove the other proteins which you can see on your gel. Which method or methods to use will depend on the properties of your protein of interest and what is available to you.
Dear Siva Palepu
as Samuel Davis the ammoniun sulfate is just a pre-purification screening.
To perform a better purification of a non-tagged protein you can perfrom a first cationic or anionic exchange (on the basis of the pI of your protein) followed from a 2nd SEC step if necessary.
best
Manuele
Ammonium sulphate precipitation is charge related so possibly size exclusion chromatography to remove salts and hopefully some of the non specific material then maybe isoelectric focussing to remove more contaminants and also concentrate the material that you do want
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