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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 1 min.

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[Bio101] Silver Staining

Biochemistry > Protein > Electrophoresis

[Abstract] 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 1 min.

Materials and Reagents

  1. 99.5 % ethanol (Sigma)
  2. 100% acetic acid (Sigma)
  3. Sodium dithionite (Sigma)
  4. Silver nitrate (Sigma)
  5. Formaldehyde solution (37 %) (Sigma)
  6. Sodium carbonate (Sigma)
  7. Sodium thiosulphate (Sigma)
  8. EDTA (Sigma)

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: 10% acetic acid (160 ml : 50 ml : 290 ml H2O).
  2. 30% ethanol.
  3. Sensitizer solution: sodium dithionite 25 mg/100 ml (sensitizer, always make it fresh)
  4. 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).
  5. 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.
    Do not use more formaldehyde then in therecipe, it might produce background. For better result, use fresh stock solution of Na2S2O3(4 mg/ml).


How to cite this protocol: Aboulaich, N. (2011). Silver Staining. Bio-protocol Bio101: e26. DOI: 10.21769/BioProtoc.26; Full Text



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