Ammonium sulfate precipitaion problem - General Methods (Apr/15/2002 )
I cannot fractionate transgene protein from plant protein by ammonium sulfate precipitation. The total protein is extracted from leave by TCA precipitation. In order to identify the protein of interest, the western analysis is performed. I can see the expected protein ban but it is hard to see. I cannot increase the amount of the protein in the SDS PAGE because the gel is overloaded. So I think ammonium sulfate can help me to get rid of the other proteins. After precipitation with 20 to 90 % saturation, about 20% of the total protein is precipitated at 20 % saturation. Finally the western analysis is performed. And the protein of interest presents in every percent saturation with the same amount. I don't understand why ammonium sulfate precipitation cannot partially purified my protein and why most of the protein precipitate at 20% saturation? Is it because of the TCA precipitation ? ( The total protein is dissolved in the buffer containing 0.06M Tris, 1 mM EDTA, 1%SDS, leupeptin and PMSF.)
Thanks in advance,
the best way out for this is reduce the volume of buffer, say 2gm tissue:1.5ml buffer this will increase the conc. to some extent.
after extraction u should first precipitate with Amonium sulphate(with required %) and then dialyse in 1:10 times of buffer. this will be the best way out
with best wishes