HPLC acetone Precipitation SDS-PAGE not Working - (Oct/06/2007 )
I would have a question. I purify my protein sample (cell membrane fraction dissolved using Triton X-100 or urea) with anion exchange chromatography (Tris/HCl, 20mM, pH=9.2). The chromatography works well and then I do precipitation of chromatography-fractions using acetone (80% final concentration, -20, overnight) and the precipitate (pellet after 20000xg, 20min) I let dry at vacuum or on air (this procedure I use always for whatever sample and it is not problem). The protein-pellet I dissolve in SDS sample buffer (for SDS PAGE). Everything seems to be fine - pellet is dissolved, samples run normally through PAGE but when I stain the gel with coomassie blue there are no bands in gel and also antibody on PVDF membrane detects almost nothing. I do not understand what is the problem? In former time this set up (working procedure) worked perfectly, but last three chromatography-acetonePrecipitation-SDSPAGE it does not work. In former time I used Tris/HCl, 20mM, pH=8,7, can it be problem? Or high concentration of salts (relatively to concentration of proteins) can be reasons that proteins do not run through gel? Should I wash pellet again with 80% acetone or acidify sample before precipitation (to have pH=7 or 8)?
Thanks for help,
Thanks for help,
may be the yield of protein is less than expected; try to stain your gel after running with silver or your blot with india ink (before blocking or if blocked with non-proteinous blocking agent); run all fractions of elution to monitor amount and pattern of eluted proteins;
high salt could be a problem but during precipitation you should have lost bulk salt