Silver Staining Procedure
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Make up following solutions:
(maybe stored for months at room temp.) Ratios are v/v.
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Fixative: |
40% methanol; 10% acetic acid; 50% water |
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Wash Sol.: |
30% ethanol in water |
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Water: |
d.i. water |
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Reductant: |
200 mg sodium thiosulfate in 1 liter water |
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Silver stain: |
2 g silver nitrate; 200 uL formaldehyde in 1 liter of water. Wrap bottle in aluminum foil to prevent light from getting in. |
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Developer: |
30 g sodium carbonate; 5 mg sodium thiosulfate; 500 uL formaldehyde in 1 L water |
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Stop Sol.: |
5% acetic acid in water |
Begin staining (Procedure is for mini-gel; takes about 2 hours; use longer incubation times for thicker gels)
- Put your gel in a large glass tray with about 1 liter volume capacity; use enough of each solution at each step to cover gel; agitate gently during steps; only touch gel with a glass rod or not at all until staining is finished; use large amounts of water for each washing step to give clean results!
- Fix proteins in gel using fixative for 20 minutes (gel can be left in here overnight etc?
- Wash three times in Wash Sol. (about 20 minutes each)
- Reduce in Reductant for 1 minute
- Wash three times in water (about 30 seconds each)
- Stain with Silver Stain for 20 minutes
- Wash three times in water (about 30 seconds each)
- Develop in Developer until bands become visible
- Wash three times in water (about 30 seconds each)
- Stop in Stop Sol. (Bands may fade if left in this solution; dry gels or save in sealed plastic bags at four degrees)
