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Silver Staining Protocol

In-Gel Staining Protocols

Silver Staining (taken from Shevchenko et al. 1996, Analytical Chemistry 68: 850)

  1. Fix gel in 50% methanol, 5% acetic acid in water for 20 min.
  2. Wash for 10 min with 50% methanol in water.
  3. Wash for 10 min with water.
  4. Sensitize the gel by treating for 1 min incubation in 0.02% sodium thiosulfate.
  5. Rinse with two changes of distilled water for 1 minute each.
  6. Submerge the gel in chilled 0.1% silver nitrate solution and agitate for 20 minutes at 4 °C.
  7. Rinse the stained gel twice with water for 1 minute each.
  8. Develop the gel in 0.04% formalin (35% Formaldehyde in water, Merck) in 2% sodium carbonate with intensive shaking.
  9. Replace with fresh developer once the developer has turned yellow.
  10. Do not over develop the gels, stop the reaction in 1% acetic acid and store in acetic acid solution until analyzed.

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