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silver staining of ip sample - (Apr/22/2011 )

hi
i am doing pull down using ip method and want to identify the proteins by mass spec.
whenever i do silver staining of the gel i have to develop it for more time as bands are visible and in turn the whole gel becomes very dark.it looks very ugly
protocol i am following is
fixation overnight-Methanol 50% Acetic acid 12%
then washing the gel with 20% ethanol for 30 minutes
wash the gel with water for 30 minutes
Sensitization (0.02% (w/v) sodium thiosulphate) - 60 min as it is a large gel
wash the gel with water for 15 minutes each -five times
Staining- Add stain solution (0.2% (w/v) silver nitrate) - 45 min
wash for 35 seconds 3 times
Developer - 6% (w/v) sodium carbonate, 0.0004% (w/v) sodium thiosulphate, 0.05% formalin) develop till bands appear it taken 15- 20 min
stop solution (12%acetic acid).
please suggest something as i have so many gels to stain

-beny-

if the gel is sds-page then you may not be removing enough sds from the gel. we use 10% ethanol, 5% acetic acid and wash 3 times. wash more times with water to remove residual acetic acid (any residual acid will react with the thiosulfate).

if you are not running sds-page then you don't need the ethanol wash but wash more times with water.

-mdfenko-