I performed protein precipitation using 40% ammonium sulphate precipitation as follows
1. 5.5 grams of ammonium sulphate salt added very slowly over a 30minutes time with gentle stirring for a 25ml of protein solution. (40% saturation) on ice
2. The solution is kept on ice with stirring for 6 hours.
3. The precipitate is pelleted down with ultracentrifuge.
4. The SDS gel of the pellet shows strong protein band at the expected position (37kDa)
I then increase the buffer quantity to 100ml which is more than what I had initially started with (25ml) which means the salt concentration is way less than 40%.
Upon dilution it dissolves i.e. I can pass the solution through a 0.22micron filter without problem which means that the protein is no more a precipitate in the solution ...but when I try to concentrate the solution using a 10kDa filter after 20 minutes of spinning at 4000rpm the solution in the centricon starts showing precipitates....which means the salt is simply not moving out of the centricon.
Even after several washes (5 to 6 washes)the protein simply seems to remain as precipitate in the centricon.
what does this mean? What else can I do to remove the salt from my protein solution?
Thank you very much for your time