Messy Western blots

Sherilyn Bell 72650.1760 at CompuServe.COM
Sun Sep 10 21:18:25 EST 1995

For some months I have been trying to examine the protein product 
of a DNA construct by transfecting cos-1 cells, lysing it in SDS 
sample buffer, running it on 8% acrylamide, blotting on 
nitrocellulose and incubating in anitbody against a portion of 
the construct.  I have been detecting with ECL but my blots have 
many nonspecific bands.  I have tried preclearing with 
untransfected cos-1 lysate, varying incubation times, washing for 
longer times and more changes (6x5min).  I have been seeing the 
product I want, but its hard to interpret with so much 
background.  The antibody is know to be specific against larger 
constructs containing the same sequence with less background.

Does anyone have suggestions?

Sherilyn Bell
slbell at

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