I am using a biotinylated probe for in-vivo biotinylation of target proteins in HeLa cells. My initial experiments consist of pulldown of biotinylated targets using Pierce's streptavidin agarose resin followed by boiling in SB and SDS WB using Vector's ABC kit (HRP-based streptavidin detection). The ultimate goal is to identify the proteins in the pulldown using MS.
The problem is this: when I load 5% of my lysate (treated vs. untreated HeLa cells lysed in 0.5% NP-40), I see a definite enhancement of biotinylated proteins in the treated sample (over a faint background of endogenously biotinylated proteins). However, the pulldown results of both samples seem identical; there is an extreme enrichment of biotinylated proteins of all MWs and I do not detect the same pattern of proteins I see in the 5% total treated lane. So, my questions are as follows: - Why might I get such different results between the total and pulldown? - Does anybody know of a way to reduce this background signal? Perhaps a cell line with less endogenously biotinylated proteins? _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
