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No band after WB revelation - (Aug/23/2010 )

Hi everybody,
Im doing wb using a HRP coupled secondary antibody and my problem is that when I put the WEST PICCO reagents I actually see brown bands on my PVDF membrane (these are the bands expected) but when Im revealing my membrane in the dark room I dont see anything on my film....!!!!! No one in my entire lab has ever seen a problem like that!!!
Any idea????

ps: I changed the film, the west picco reagents, and the revelation liquid...nothing!

Please your help is really needed.
Thank you.


Your bands are too strong. The HRP signal is burning out before you get it to imaging. That's what the brown spots are. There should be a section in the Pico kit instructions for troubleshooting brown spots on the membrane.


yeah fishdoc is right. Try to dilute the reagents or use other ones less sensitive