Protein silver staining.
Stephen R. Decker
Sdecker at Vines.colostate.edu
Tue Mar 5 17:24:17 EST 1996
The following is the silver stain protocol from the Hoefer protein electrophoresis
manual (summarized, of course):
All steps should be about 100 mL for a mini gel, and gently swirled at RT.
1- 40% MeOH, 7% Acetic acid 30 mins
rinse with dH2O
2- 7% Acetic acid, 5% meOH 30 mins
3- 10% glutaraldehyde, 30 mins
4- dH2O wash, running at least 2 hours, longer for clearer background
can go O/N w/o problem
5- 5ug/mL DTT for 30 mins, do not rinse, drain well
6- 0.1%(w/v) AgNO3, 30 mins
rinse quickly in dH2O, then with two small batches of developer
7- 3% Na2CO3, 0.019% formaldehyde
This is the developer and should be made just prior to using. All other
reagents can be stored at RT for at least a month, with the exception of
glutaraldehyde which needs to be in the dark at 4oC. Add the developer and swirl
until just underdeveloped. Drain and add solution from step 2 to stop rxn. Can be
stored in this solution. This is the trickiest step because the developing rxn
continues until the acid from the stop bath gets into the gel.
If yo are in a hurry to get the gel, you can cut the first two steps back to 15 mins
each and the long wash to about an hour, if you don't mind a little background.
More information about the Proteins