High background with FLAG antibody - (Mar/02/2009 )
I had initially tried detecting using my ATP1B1 primary antibody followed by antimouse HRP secondary antibody, but this gave no signal. I then did a dot blot using FLAG primary antibody and antimyc HRP secondary antibody on the same samples. The dot blot did have lots of background for FLAG but i could see the "dot". Dot blot for the beta actin Ab somehow didnt show up. I applied the same conditions for the initial blot after stripping. I have nice bands for my control beta actin primary Ab, antimouse secondary Ab. But for the FLAG Ab, i see a big blob in the shape of my blot.
try changing your blocking conditions, and/or shortening the time you have your secondary incubating for.