High Background in western Blotting - (Apr/11/2007 )
I am doing western blotting for some time and awaiting for good and clean results.Due to higher background i m not able to do ECL for detection,i toggled with all options like change in abs (Primary and Secondary)dilution ,membrane and buffer.But recently i find that band is appearing with dye front as well,therefore i think that reason might be SDS,so i removed SDS from transfer buffer by today and expecting positive. I am waiting for suggestions which can enrich my experience.
we need much more details about your protocol to help you. especially the concentration of Ab, the blocking buffer, incubation buffer, and maybe a picture of your blot.
it could be plenty of things
Remember, that some antibodies ( primary) are not very specific, and one will always get background.
But one has to try reducing the antibody conc. and blocking for longer or with different blocking agents.