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University of Wisconsin–Madison

Mass Spectrometry / Proteomics Facility

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Non-Destructive Silver Staining (modified for MS)


  • Fix the gel for at least an hour by gently shaking in 40% Ethanol [EtOH]/10% Acetic Acid [HAc]/50% Millipore water (200ml or about five times gel volume).

Washing and Sensitizing

  • Wash the gel two (2) times for 20 minutes each in 30% EtOH (same volume requirements as above).
  • Wash the gel once with Millipore water for 20 minutes
  • Sensitize the gel in 0.02% Sodium Thiosulfate [Na2S2O3] for 1 minute.
  • Wash the gel three (3) times for 30 seconds with Millipore water.

Staining and Developing

  • Incubate the gel for 20 minutes with cold 0.1% Silver Nitrate [AgNO3]/0.02% formal [37% formaldehyde] solution.
  • Wash the gel four (4) times for 30 seconds with Millipore water (room temperature).
  • Develop the gel in 3% Sodium Carbonate [Na2CO3]/0.05% formalin, observe the color and change solution when developer turns yellow. Stop the reaction when staining is sufficient.

Terminating Staining and Storage

  • Wash the gel with Millipore water for 30 seconds.
  • Terminate development by incubating in 5% HAc for 5 minutes.
  • Wash the gel three (3) times for 10 minutes with Millipore water.
  • If gel is to be stored for longer time, keep immersed in 1% HAc at 4C.


  • Blum et al. Electrophoresis. 1987; 8: 93-99.