Silver staining problem

[hericorreia]

Hi everyone. I have a problem staining an second dimentional gel of proteins. I get negative spots after stain with silver nitrate. I had done different silver staining protocols and the result is always the same. negative spots and dark baground. I have changed the reagents, wash steps, incubation times with fix solution and silver nitrate, protein concentrations- etc.
Have you any suggestions to resolve this trouble?
thank you

[JackBean]

didn't you overstain? Did you try to decrease the last time, before you stop the staining with acetic acid? (sorry, I don't remember these photographic terms). Do you control, how long you have it in that solution or you just have it there for designed time?

[canalon]

I've had that in a few occasion on simple gel where I had too much protein (intense coomassie staining), the silver stain gave a blank center with only the side lightly colored. But I never had a negative stain on the whole gel. But I use a Bio Rad kit, rather than home made reagent.
Sorry not much help here. Maybe you should try Coomassie staining ? that is what is recommended by the lab I plan to use for sequencing my protein if I finally manage to express enough of it.
Sample protocols available here:
http://www.sickkids.ca/Research/APTC/Ma ... index.html