Berglund:Silverstaining
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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
- Fix gel with destaining solution for 20-30 minutes
- Rinse gel with water for 20-60 min to remove acid
- Sensitize gel with 0.02% sodium thiosulfate for 1-2 min
- discard solution and rinse gel with two changes of water (1 minute each)
- incubate gel in chilled 0.1% silver solution for 20-40 minutes at RT
- discard solution and rinse gel with two changes of water (1 minute each)
- Develop gel with Developing solution with shaking (replace developing solution when it turns yellow)
- Quench when sufficient staining is obtained by discarding developing solution and adding quench solution
- store in 1% acetic acid at 4°C or dry gel
Notes
- don't touch gel with bare fingers, wash latex gloves before handing gels, use dishes that are only for silverstaining
- pressure by forceps on the gel can leave marks that will be seen by the silver stain
- you can silver stain and then coomassie stain the gel, I don't know if you can do the reverse
- this method should allow you to mass spec proteins from the gel after silverstaining
- overloading of protein on the gel can result in bands that are brown on the outside and white in the middle
- 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
- do not use glutaraldehyde as the sensitizing agent- it is also a protein crosslinking agent