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Western blot doesn't work anymore - low sensitivty in Western blots (Jan/28/2003 )

Could anybody help me?

I do Western blots, and am now using nitrocellulose membranes (Schleier & Schuell). Somehow, although I'm using the same antibodies as before, the signal is most of the time very low, even with extended illumination times. We've been testing through various types of blocking reagents (dry milk, casein,...) so that's unlikely to be the cause of the problem. Could it be the type of membrane we are using. I had good experiences in the past with PVDF.


Both PVDF and nitrocellulose have worked well for me in the past, and I've seen similar results from each. What sort of substrate and buffer system are you using?
What kind of concentrations are you using?

-Tim Dwyer-