KO mice show band for knocked-out protein - (May/25/2011 )

I have been trying to optimize my Western Blot for about 3 months now. I have a mouse strain that has a gene for a 60kDa protein knocked out, but when I run these westerns, I see it is expressed in both the WT (or HE) and KO mice. Sometimes, the blots show lots of non-specific banding. The attached blot is the most recent one that only shows non-specific banding just below the 70kDA mark (). The lighter band at 60kDa is (or should be) my protein of interest. Strangely, the blots work best when I load over 100ug total protein. Can anyone give me some tips/explanations on why this might be happening? The postdoc in my lab before I started working here go the correct results, although they were not publishable quality, so this should be working. As best I can tell, I am following his protocol exactly.

Thank you for the help!

You should test the antibody by peptide competition to prove that the band you are seeing is actually a specific binding. At the moment your western does not look very specific, and with over 100ug of protein loaded I would expect brighter bands in the WT lanes. You should also include a positive control of some sort - perhaps cultured cells that express the protein.