Silver Staining Protocol
Silver Staining Protocol
Sensitivity: 5-10 ng/mm2 or ca. 1.5 ng/band
For two large 20 cm x 20 cm x 1.5 cm gels, use 500 ml per tray
Reagents needed:
ethanol
methanol
acetic acid
formaldehyde (37%)
sodium thiosulfate
silver nitrate
sodium carbonate
Procedure
1. Fix gel in 50% methanol, 12% acetic acid, 0.05% formaldehyde for 2 hrs or overnight; 250 ml: 60 ml: 0.67 ml: 190 ml, methanol: acetic acid: 37% formaldehyde: water
2. Wash gel in 35% ethanol - three times for 20 min each; 175 ml: 325 ml; ethanol: water
3. Sensitize gel in 0.02% Na2S2O3 for 2 min.; 0.1 g sodium thiosulfate per 500 ml water
4. Wash gel in water - three times for 5 min each
5. Stain gel in 0.2% AgNO3, 0.076% formaldehyde for 20 min.;
1 g silver nitrate: 1 ml 37% formaldehyde: 500 ml water
6. Wash gel in water - two times for 1 min each
7. Develop gel in 6% Na2CO3, 0.05% formaldehyde, 0.004% Na2S2O3; 30 g sodium carbonate: 0.67 ml 37% formaldehyde: 0.02 g sodium thiosulfate per 500 ml
8. Stop development in 50% methanol, 12% acetic acid for 5 min
250 ml methanol: 60 ml acetic acid: 190 ml water
9. Store gel at 4oC in 1% acetic acid
Reference
Mortz E. Krogh T N. Vorum H. Gorg A. (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1(11):1359-63.
Top of page
|
|
Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
|
