Mass Spectrometry-compatible Silver Staining of Protein Gels

  1. Wash gel 2×20 minutes (or overnight) in 50% methanol/10% acetic acid/40% FPLCgrade water. Use 50-100 mL.
  2. Wash 10 minutes in 20% ethanol.
  3. Wash 10 minutes in water.
  4. Reduce with 0.2 g/L sodium thiosulfate, 1 minute.
  5. Wash 2×20 s with water.
  6. Incubate with pre-warmed 2 g/L silver nitrate, 37°C, 30 minutes.
  7. Wash 20 s with water.
  8. Incubate with developing solution, rocking for <30 s; discard.
  9. Add fresh developing solution and incubate just until desired signal is reached.
  10. Immediately transfer to 1% acetic acid stop bath. Gels may be kept in this solution for many days.

(Note: Adapted from a protocol from the University of Toronto’s former Mass Spec Core facility)


Developing Solution (1L)
30 g Na2CO3.H2O
1.4 mL 37% formaldehyde solution
50 mL of 0.2 g/L sodium thiosulfate solution