Silver Staining Procedure

 

Make up following solutions:
(maybe stored for months at room temp.) Ratios are v/v.

Fixative:	40% methanol; 10% acetic acid; 50% water
Wash Sol.:	30% ethanol in water
Water:	d.i. water
Reductant:	200 mg sodium thiosulfate in 1 liter water
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.
Developer:	30 g sodium carbonate; 5 mg sodium thiosulfate; 500 uL formaldehyde in 1 L water
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)

1. 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!
2. Fix proteins in gel using fixative for 20 minutes (gel can be left in here overnight etc?
3. Wash three times in Wash Sol. (about 20 minutes each)
4. Reduce in Reductant for 1 minute
5. Wash three times in water (about 30 seconds each)
6. Stain with Silver Stain for 20 minutes
7. Wash three times in water (about 30 seconds each)
8. Develop in Developer until bands become visible
9. Wash three times in water (about 30 seconds each)
10. Stop in Stop Sol. (Bands may fade if left in this solution; dry gels or save in sealed plastic bags at four degrees)