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Silver Staining of Protein Gels (silver diammine)

Add 2.5 mM Sodium Bisulfate to running buffer before running PAGEgelTM (To 500 ml running buffer, add 1.3 g Sodium Bisulfate).

Solutions: Fix: 40% MeOH, 10% AcCOOH. Add 20 mM Sodium bisulfite (0.21 g/100 ml) before use for PAGEgel. Sensitizer: 10% Glutaraldehyde (from 25% stock; kept at 4ºC) Ammoniacal Silver (make fresh and use within 5min): Solution A: 0.8g AgNO3 in 4 ml ddH2O. Solution B: 21ml of ddH2O; 400 µl of 10 N NaOH and 1.4ml of 14.8M ammonium hydroxide (Acros #423300250, kept at 4ºC). Add Solution A dropwise into Solution B with constant stirring, then add water to 100ml. Developer (make fresh): 0.01% citric acid; 0.1% formaldehyde; 1 ppb Na2S2O4 2 ml of 1% citric acid 200 μl of 38% Formaldehyde 2 μl of 10% Na2S2O4 (keeps for a week at RT) 200ml ddH2O Stop: 2% AcCOOH, 20% EtOH

Protocol -All incubations are carried out with gentle shaking on a reciprocal shaking platform. -All steps take place in a clean plastic box or glass dish. 15cm TC dish works great. -Steps in grey are optional.

  • If point streaking becomes an issue (“rainy” lane), add 200 mM iodoacetamide (4M stock in methylformamine) to samples after boiling.

Incubate overnight in 250ml Fix. 1h will work in a pinch. Wash gel in ddH2O for 15 min. Wash gel in ICE-COLD ddH2O for 15 min. Incubate gel in cold Sensitizer for 30 min. Wash gel in ICE-COLD ddH2O for 15 min (4x). Incubate 10-30 min in Ammoniacal Silver (watch that gel does not become yellow). Wash gel in ddH2O for 5min (3x). Transfer gel to a new dish. Incubate in Developer. Replace solution as soon as it starts to get yellow/cloudy. Add Stop for no longer than 30min. Wash gel in water for >1h. Scan gel. Wash gel in drying solution of your choice and dry gel.