Berglund:Silverstaining

Adapted from Shevchenko et al, 1996, Anal. Chem. 68: 850-858 (Adapted by Protein Research Group- Dept of Mol. Bio. -Odense University- Denmark)

Materials

destaining solution- Methanol:acetic acid: water (45:5:45)
sensitizing agent 0.02% sodium thiosulfate- 0.1g Na2S2O3 per 500ml water
0.1% silver solution- 0.1g in 100ml water
Developing solution- 150µl 37% formalin solution, 12.5g Na2Co3, 500ml water
Quench solution- 5ml acetic acid in 500ml water

Methods

1. Fix gel with destaining solution for 20-30 minutes
   
2. Rinse gel with water for 20-60 min to remove acid
   
3. Sensitize gel with 0.02% sodium thiosulfate for 1-2 min
   
4. discard solution and rinse gel with two changes of water (1 minute each)
   
5. incubate gel in chilled 0.1% silver solution for 20-40 minutes at RT
   
6. discard solution and rinse gel with two changes of water (1 minute each)
   
7. Develop gel with Developing solution with shaking (replace developing solution when it turns yellow)
   
8. Quench when sufficient staining is obtained by discarding developing solution and adding quench solution
   
9. store in 1% acetic acid at 4°C or dry gel
   

Notes

1. don't touch gel with bare fingers, wash latex gloves before handing gels, use dishes that are only for silverstaining
2. pressure by forceps on the gel can leave marks that will be seen by the silver stain
3. you can silver stain and then coomassie stain the gel, I don't know if you can do the reverse
4. this method should allow you to mass spec proteins from the gel after silverstaining
5. overloading of protein on the gel can result in bands that are brown on the outside and white in the middle
6. for highest sensitivity rinse for 60 minutes, or more, after the gel has been run and fixed, this helps to keep the background transparent during development
7. do not use glutaraldehyde as the sensitizing agent- it is also a protein crosslinking agent