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Protein Precipitation

protein precip ELISA protein extraction acetone precip

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#1 Calbes

Calbes

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Posted 09 March 2012 - 08:09 AM

Hi, I am performing an ELISA with Arabidopsis tissue. After extracting the protein I add 6X volume of ice-cold acetone and place the samples in the freezer for 3 hours. I then vortex the samples, but I get 3 phases of material. A top, liquid acetone phase that I discard, a thin middle phase that looks like it might be the protein precipitate, and a 50-100 μL gel-like bottom phase. Another person in the lab who previously performed ELISA with this protocol said he did not get the bottom phase and was able to dry the precipitate with no problem. If I include the bottom phase in the rest of the assay I do not get good results. It cannot be easily separated from the thin middle phase which I believe is the precipitated protein. Does anyone have any suggestions on how to separate the middle and bottom phases? And what the bottom phase may be? My extraction buffer contains 0.5 M tris (pH 8), 1% SDS, 30% sucrose 2.4 g/L aminocaprioc acid, and 0.72 g/L benzamidine.

Thank you for you suggestions,
AL





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