Ranan, my first thought was that you possibly have some sort of protease in your prep that becomes active when the inhibitors are removed by the desalting process (as small molecules migrate slower that large molecules and you probably won't include inhibitors in the column buffers due to their large volume).
But I understand it correctly that the bands occur in *all* fractions you are collecting from the column (even in an empty run)? So, possibly that what you see on the gel / western is not protein, but some sort of leakage that comes from the column. If you don't have the opportunity to identify the nature of the band by mass spectrometry, you could do some simple experiments to get an idea what you might have; either your gel is degraded and is leaking some sort of possibly polysaccharides or there is a contamination on the column (when it has been used for something else previously or not been stored properly). You didn't write about the nature of your sample; is there a possibility that it contains enzymes that may degrade the sepharose? a) try to thoroughly clean the column as described in the manual. If the problem persists, b) try to digest the eluate with proteases, eg trypsin. if there is protein, it should become degraded c) add some (sodium) metaperiodate in order to oxidize diols. If your band gets degraded, it is a polysachharide. You also might simply ask the technical service of your supplier for help. They should know their stuff. Other possibilities are to snatch a different batch from the neighbor lab and check if it behaves the same. HTH Wo PS is gel filtration the first step of your purification process? _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
