Search

Silver Staining   

Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

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).
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aboulaich, N. (2011). Silver Staining. Bio-protocol Bio101: e26. DOI: 10.21769/BioProtoc.26.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.