Protocol for Silver Staining

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 venders, please follow their specific instructions.

Fixation

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.