Using the antibody linked in the end, I still get at least three non-specific bands in astrocyte lysates. Does anybody have the same problem?
http://www.sigmaaldrich.com/catalog/product/sigma/n9657
I don't have problem with this specific antibody but with a variety of antibodies against plasma proteins: multiple non-specific bands coming from the secondary anti-IgG-HRP. My solution was to custom-couple my primary antibodies directly to HRP, and the problems were all gone.
If your sample is from tissue some of the bands might be Ig chains (mouse Ig and anti-mouse Ig)
Check whether the 2o Antibody cross react with the sample without 1o antibody at different dilutions. Try using different 2o antibody.
Titrate optimal 1o Ab concentration to get optimal signal.
Most anti-IgG-HRP will cross react with IgGs from other species. Personally speaking, your time will be better spent in conjugating HRP directly to your Ab (provided you have enough primary, e.g., 100 ug). I conjugated dozens of anti-plasma protein Abs, and found them all work beautifully at 1:5000 and saved my time in only one step Ab incubation.
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