Discrepance between peaks in chromatogram and amount of proteins? - (Feb/05/2008 )
I have the following problem:
I do anion exchange chromatography - pH=9.2 (Tris-HCl), 0-28min, 0-70% (then elution with 100%) of 1M NaCl, detection at 280nm. The highest and sharpest peaks in the chromatogram are comming between 5.-14. min. All fractions (or aliquot of them) I precipite with acetone (80% final concentration). Then I dissolve protein pellet in SDS PAGE loading buffer a I do western blot analysis of all fractions. But surprisingly, when I detect proteins with antibody-HRP-ECL system on PVDF membrane and when I do coomassie blue staining of those gels most of the proteins are in fractions of 15.-28. (I can clearly see many bands in gel) and almost nothing I can see in fractions of 5.-14. (where actually I have the highest peaks at 280nm). Also the protein pellet after acetone precipititation in fractions of 5.-14. is not very big. TCA precipitation does not work better.
What can be problem? The peaks are not proteins (I use for example crude cytosolic fraction for AEC)?
The proteins in the first several fractions are probably quite basic, can it influence acetone precipitation or dissolving in SDS loading buffer?
Thanks for suggestions,
try running the gel with aliquots of the fraction without precipitating. don't worry about the salt for this test. it will help you see what you are losing when you precipitate.