Messy Western blots

Ri-Ya Yang/SBR4/4-9980 ryang at SCRIPPS.EDU
Mon Sep 11 10:55:10 EST 1995


Maybe the the antibody concentrations were too high.

Ri-Yao


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
> 
> 



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