Protein silver staining.

Stephen R. Decker Sdecker at Vines.colostate.edu
Tue Mar 5 17:24:17 EST 1996


Belinda,

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
      rinse
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.

Good Luck

Steve




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