I'm an MD new to bench research. I am working with a protein which has pretty terrible antibodies, at least for western blotting. I've tried several different antibodies with variable results. The problem is there is so much nonspecific stuff that it makes it very hard to detect my protein of interest.

What techniques have you had success with to cut down on nonspecific bands? I've started blocking anywhere from 4 hours to overnight with modest improvement. Someone suggested harvesting the cells with trypsin rather than scraping the plate. Anyone else had luck doing it this way? Is there other steps in the western that could help with this? Would going down on the secondary antibody concentration (currently using mouse 1:5000) help?

I appreciate any assistance,

Ken