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 sickkids.on.ca



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