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Silver Staining   

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Materials and Reagents

  1. 99.5 % ethanol (Sigma-Aldrich )
  2. Silver nitrate (AgNO3) (Sigma-Aldrich )
  3. Formaldehyde solution (37 %) (Sigma-Aldrich)
  4. Sodium carbonate (Sigma-Aldrich)
  5. Sodium thiosulphate (Sigma-Aldrich)
  6. EDTA (Sigma-Aldrich)
  7. Sodium dithionite (Sigma-Aldrich)
  8. 30% ethanol
  9. Acetic acid
  10. Stop solution (see Recipes)
  11. Fixation solution (see Recipes)
  12. Sensitizer solution (see Recipes)
  13. Staining solution (see Recipes)
  14. Developer solution (see Recipes)

Equipment

  1. Shaker

Procedure

  1. Add fixation solution on gel and incubate overnight or minimum 2 h with gentle shaking.
  2. Wash gel with gentle shaking with 30% ethanol 3 X 10 min.
  3. Wash gel with dest. water 2 X 10 min.
  4. Incubate gel with sensitizer solution for 1 min.
  5. Wash gel with dest. water 2 X 1min.
  6. Incubate gel with staining solution for 25 min.
  7. Wash gel with dest. water for 1 min.
  8. Add developer solution (about 2-3 min) with gentle mixing.
  9. Add stop solution before the protein bands get too dark stained.

Recipes

  1. Fixation solution
    30% ethanol:        160 ml
    10% acetic acid     50 ml
    H2O                        290 ml
  2. Sensitizer solution
    Sodium dithionite 25 mg/100 ml (sensitizer, always make it fresh)
  3. Staining solution
    0.2% AgNO3 (400 μl/40 ml from 20% AgNO3 stock solution from the freezer)
    Careful, it is strong oxidizer and blackens everything)
    + 3 μl/40 ml Formaldehyde solution (36-38%, must be fresh)
  4. Developer solutiuon
    6% sodium carbonate (3 g/50 ml)
    4 μg/ml sodium thiosulfate (Na2S2O3, 50 μl of stock solution 4 mg/ml, can be stored a few weeks in the fridge)
    + formaldehyde 25 μl/50 ml. Always make developer fresh
    Note: Do not use more formaldehyde then in therecipe, it might produce background. For better result, use fresh stock solution of Na2S2O3(4 mg/ml).
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aboulaich, N. (2011). Silver Staining. Bio-101: e26. DOI: 10.21769/BioProtoc.26.
Q&A
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Ye Liu
Van Andel Institute
May I know the recipe for the stop solution?
3/6/2020 7:14:03 AM Reply
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