Problem with endogenously biotinylated proteins

Barbara MacGregor via methods%40net.bio.net (by bmacgreg from unc.edu)
Sat Dec 26 10:54:46 EST 2009


Hi Ranen,

This might not be feasible in your system - but there are photocleavable
biotins available for attachment to probes. After streptavidin capture,
target proteins could be released by exposure to light, while naturally
biotinylated proteins would remain bound.

Barbara MacGregor


From: ranen aviner <ranen.aviner from gmail.com>
Date: December 23, 2009 2:07:17 AM EST
To: methods from magpie.bio.indiana.edu
Subject: Problem with endogenously biotinylated proteins


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?



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