Bioanalytical Research

Bioanalytical Research Group

Silver Staining of Polyacrylamide Gels

1. Fix gel with Destaining solution.

 • MeOH : acetic acid : water ( 45 : 5 : 45 )

 • Time: 20 - 30 min.

2. Rinse gel with water ( 20 - 60 min. ) to remove acid.

3. Sensitize gel with 0.02 % sodium thiosulfate

 • 0.1g Na2S2O3 per 0.5l water

 • Time: 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

 • 0.5g AgNO3 per 0.5l water

 • Time: 30 min.

6. Discard solution and rinse gel with two changes of water (1 minute each)

7. Develop gel with Developing solution (0.04% formaldehyde; 2% Na2CO3):

 • 12.5g Na2CO3

 • 150µl 37% formaldehyde solution

 • 0.5l water

 • Shaking table

 • Important: Replace developing solution when it turns yellow

8. Quench development when sufficient staining is obtained: Discard solution and add 1% acetic acid

 • 5ml acetic acid per 0.5l water

9. Store the silver stained gel in 1% acetic acid solution at 4oC.

Comments ( by Andrej Shevchenko): 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!

Reference

Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels Shevchenko A., Wilm, M., Vorm, O. and Mann, M. Anal. Chem. 68, 850-858 (1996).