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High Background in western Blotting - (Apr/11/2007 )

ph34r.gif 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.

-Harsh Vardhan-

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.