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No band after WB revelation


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#1 Leen

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Posted 23 August 2010 - 01:46 AM

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.

#2 fishdoc

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Posted 23 August 2010 - 04:57 AM

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.

#3 laurequillo

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Posted 23 August 2010 - 05:03 AM

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