Silver Staining Protocol

Silver Staining Protocol

Silver staining is less compatible with the mass spectrometric analysis. Therefore, we don't recommend using silver staining for any samples that will subsequently be submitted for MS analysis. If you can not visualize bands with coomasie and you are not able to scale up your isolation of protein to reach coomasie stainable levels, you must use a mass spec compatible silver stain protocol.

  • You must only use methanol and acetic acid during the fixing step.
  • You must not use any solutions containing formaldehyde or glutaraldehyde to fix the gel.
  • You should use 1.0 to 1.5mm gels and only stain the gel long enough (usually only a few minutes) to detect the bands of interest.
  • You should clean a glass or plastic tray with detergent and rinse it thoroughly.
  • You should wear clean, disposable gloves and never touch the gel bands directly.

The following is a protocol used at the proteomics resource center for Silver staining. If you are using Silver staining kit from Sigma, Invitrogen, or other vendors, please follow their specific instructions.


  1. Fix gel in 150 ml 50% methanol + 5% acetic acid for 20 min.
  2. Wash in 150 ml 50% methanol for 10 min.
  3. Wash in water for 10 min.

    For 1 gel, mix 75 ml methanol, 7.5 ml acetic acid, and 67.5 ml water.


  4. Incubate with 150 ml 0thiosulfate for 1 min.
  5. Rinse with water for 1 min. 2X.

    0.02% Sodium Thiosulfate: For 1 gel, add 30mg sodium thiosulfate-5 hydrate to 150ml water.

    Silver reaction

  6. Submerge gel in 150ml 0.1% silver nitrate with 0.08% formalin (37%) for 20 min.
  7. Rinse with water for 1 min. 2X.

    0.1% Silver Nitrate and 0.08% Formalin (37%): For 1 gel, add 150 mg silver nitrate and 120ul 37% formalin to 150ml water.

    Developing: Must make fresh solution

  8. Incubate with 150ml 2% sodium carbonate with 0.04% formalin (37%) until desired intensity of staining occurs. If developer turns yellow, which it often does within 30 sec, then discard and replace with fresh 150ml developer.

    2% sodium carbonate + 0.04% Formalin: For 1 gel, add 6g sodium carbonate to 300ml water. Just prior to use, add 120ul 37% Formaldehyde to the 300ml.


  9. Wash gel in 150ml 5% acetic acid for 10 min.

    5% acetic acid: For 1 gel, add 7.5 ml acetic acid to 150ml water.


  10. Wash gel in water for 5 min.

    Permanent Storage

  11. Incubate gel in 150ml preserving solution for 20min.

    Preserving solution: For 1 gel, add 13.2ml glycerol (100% w/w) to 150 ml water.