Silver Staining Protocol for SDS-PAGE Gels (Mass Spectrometry compatible)
Approximate time required: 2.5 hours
Reagents Required (Electrophoresis grade or higher):
§ Ethanol (reagent grade 95%)
§ Glacial acetic acid
§ Milli-Q H2O (or equivalent)
§ Sodium thiosulphate (pentahydrate)
§ Sodium acetate (anhydrous)
§ Silver nitrate
§ Sodium Carbonate (anhydrous)
§ Formaldehyde (37% w/v)
§ EDTA-Na2 (0.5M)
Note:
§ Always use freshly prepared solutions
§ Perform all steps at 21-25°C with constant gentle agitation
§ Silver stained gel may be stored in fixation solution
Protocol:
1. Double Fixation 30 minutes x 2
(or 30 min+ O/N)
§ Ethanol: 100mL
§ Glacial acetic acid: 25mL
§ Make up to 250mL with Milli-Q water
2. Sensitizing 30 minutes
§ Ethanol: 37.5mL
§ Sodium thiosulphate (0.5g/10mL): 5mL
§ Sodium acetate: 8.5g
§ Make up to 125mL with Milli-Q water
3. Washing 5 minutes x 3
§ Milli-Q water
4. Silver Reaction 20 minutes
§ Silver nitrate (0.35g/14mL): 12.5mL
§ Make up to 125mL with Milli-Q water
5. Washing 1 minute x 2
§ Milli-Q water
6. Developing 2 ~10 minutes
§ Sodium carbonate: 3.125g
§ Formaldehyde (37% w/v): 0.025mL
§ Make up to 125mL with Milli-Q water
7. Stopping 10 minutes
§ EDTA (0.5M): 10mL
§ Make up to 125mL with Milli-Q water
8. Washing 5 minutes x 3
§ Milli-Q water
9. Destaining
Silver-stained proteins are destained with chemical reducers to remove the silver as described previously with the following critical modifications: The reactive substances of the chemical reducers are potassium ferricyanide and sodium thiosulfate. These chemical agents were prepared prior to digestion as two stock solutions of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate, both dissolved in water. A working solution was prepared by mixing a 1:1 ratio of the above stock solutions. Note that this working solution is unstable, and therefore should be prepared fresh for each reaction. Thirty to fifty microliters of working solution were added to cover the gel bands and occasionally vortexed. The stain intensity was monitored until the brownish color disappeared, then the gel band was rinsed a few times with water to stop the reaction. Next, 200 mM ammonium bicarbonate was added to cover the gel for 20 min and was then discarded. Subsequently, the gel piece was cut into small cubes, washed with water.