Bioruffo
Molecular Biology protocols, utilities and oddities
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Description
 
This is a very fast and convenient silver staining protocol for SDS-PAGE minigels. If you run two identical PAGE gels, it can be conveniently completed on one gel while the other is being blotted to the membrane.
This protocol origins from the paper "A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels" by M. V. Nesterenko, M. Tilley, and S. J. Upton (J Biochem Biophys Methods 1994;28:239-242). This protocol is very similar to the original, with the following differences.
 
The original protocol in Nesterenko et al.:
- used 60 ml of each solution for minigels (I use 30ml)
- included three 5sec rinses after the wash at step 3
- step 5 (pretreatment in thiosulphate) was for 1 min
- step 8 included only 2 rinses
- step 9 would include 12.5 ul of formaldehyde instead of 12 ul.

 
Reagent stock solutions:
 
Acetone Stock:
   50% Acetone in dH2O
 
TCA Stock:
   50% Trichloroacetic acid in dH2O

Formaldehyde Stock:
   37% formaldehyde
 
Na2SO3 Stock:
   10% sodium thiosulphate in dH2O (stable for 4 months)
 
AgNO3 Stock:
   20% silver nitrate in dH2O (store in dark for up to 4 months)
 
 
Work solutions should be prepared fresh from the stocks.
 
 
Protocol 
 
Quantities and times are for minigels.
 
All steps are at RT.
 
1. Fix, 5'
   30 ml Acetone_Stock
   + 750 ul TCA_Stock
   + 12.5 ul Formaldehyde_Stock
 
2. Rinse, 3 times w/ dH2O (30-50 ml each)
 
3. Wash, 5' 
   30 ml dH2O
 
4. Pretreatment, 5'
   30 ml Acetone_Stock
 
5. Pretreatment 5' 
   30 ml H2O
   + 50 ul Na2SO3_Stock
 
6. Rinse 3 times w/ dH2O (30-50 ml each)
 
7. Impregnate, 8'
   30 ml H2O
   + 300 ul Formaldehyde_Stock
   + 400 ul AgNO3_Stock
         (if a slight precipitate appears, mix to dissolve)
 
8. Rinse 5 times w/dH2O (30-50 ml each)
 
9. Development (some seconds) 
   30 ml H2O
   + 600 mg Na2CO3 (disodium carbonate)
   + 12 ul Formaldehyde_Stock
   + 12.5 ul Na2SO3_Stock
 
10. Stop 
   30 ml H2O
   + 300 ul Acetic Acid (glacial)