Messy Western blots
ryang at SCRIPPS.EDU
Mon Sep 11 10:55:10 EST 1995
Maybe the the antibody concentrations were too high.
On 11 Sep 1995, Sherilyn Bell wrote:
> 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
More information about the Proteins