I use a silver staining kit from GE Healthcare for 2D electrophoresis and IEF. But according to the handbook the procedures are different for each electrophoresis. For example acetic acid is used to fix 2D gels and TCA is used to fix IEF gel. Why?

Another problem is, a black line always forms on my gel after staining, on the spot where I put my acidic electrode strip. Any suggestions?

Thanks a lot.

TCA used to fix IEF, I think it has something to do with Ampholine, which supposed to be removed for silver staining.

for the black line, your acidic electrode strip maybe too acidic

there are several good silver staining protocol in the PROTOCOL, and I have posted the one I am using daily, which works perfect for me
http://www.scientistsolutions.com/index.php?a=protocol&c=10&p=704

Thanks for the answer.
Do you also know what indicates overloading? I can't get a band of my target protein on my IEF gel. I always see the white precipitate at the expected spot after fixation step, but at the end of the staining no band appears. The marker bands are there though.

loading for silver staining of 2D gel varies from 1-100ug protein/IEF stripe, mainly depend on your sample type (how many lines on the 2nd dimension, and how many proteins per line). I will start with 20-40.

you do not need to silver stain you IEF, CBB stain is good enough. the loading in IEF is too much for silver staining, and it may probably give you too dark background or negative bands.

and a prolonged fixation will always be helpful in silver staining

Thanks. I will try your advice and let you know if it works. :)