Silver Staining of PAGE Gels
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Immediately after the electrophoretic run is terminated, slice the stacking gel from the slab, and place the gel in a glass dish with 200 ml (8mm x 7mm mini-gel) of 40% methanol/10% acetic acid for the length of time indicated in the table below. For convenience, the gel can remain in this first fixative indefinitely. Acetic acid is optional in all fixatives if staining nucleic acids.
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Follow the steps in sequence 1-13 as indicated in the following table. Use of a rotary or rocker platform is recommended. All steps are performed at room temperature. The oxidizer and silver reagent are supplied as 10x concentrates, and are diluted 1:10 in dH2O just before use. Store the concentrates at 4°C.
Duration <0.5 mm 0.5-1.0 mm >1.0 mm Reagent Volume a gel gel gel 1. Fixative I 400 ml 30 min 30 min 60 min 2. Fixative II 400 ml 15 min 15 min 30 min 3. Fixative II 400 ml 15 min 15 min 30 min 4. Oxidizer 200 ml 3 min 5 min 10 min 5. dH2O 400 ml -- 5 min 10 min 6. dH2O 400 ml -- 5 min 10 min 7. dH2O 400 ml -- -- 10 min 8. Silver reagent 200 ml 15 min 20 min 30 min 9. dH2O 400 ml -- 1 min 2 min 10. Developer 200 ml 30 sec. Develop until solution turns yellow or until a brown "smoky" precipitate appears. Then pour off developer, and add fresh developer. 11. Developer 200 ml 5 min 5 min 5 min 12. Developer 200 ml -- 5 min 5 min 13. Stop solution 400 ml 5 min 5 min 5 min -- Indicates that the step is omitted. a Recommended volumes for 16 cm slab gel in a 21 x 21 x 5 cm baking dish. For smaller gels in a smaller container, decrease volumes proportionately. For 8 mm x 7 mm mini-gels, use one-half the volume shown.
Fixative I (500 ml) Fixative II (500 ml) methanol 200.0 ml (40%) ethanol 50.0 ml (10%) acetic acid 50.0 ml (10%) acetic acid 25.0 ml (5%) dH2O 250.0 ml dH2O 425.0 ml
Stop Solution (500 ml) Kit Reagents acetic acid 25.0 ml (5%) 1. Oxidizer: Dilute 1:10 with dH2O. dH2O 475.0 ml 2. Silver reagent: Dilute 1:10 with dH2O. 3. Developer: Dissolve 16 g per 500 ml dH2O.
Caution: All the kit reagents are poisonous. The oxidizer contains potassium dichromate and nitric acid, the silver reagent contains silver nitrate, and the developer contains sodium carbonate and paraformaldehyde. Wear gloves and lab coat when handling, do not mouth pipette.
