Biochemistry

scientificprotocols authored almost 4 years ago

Authors: T Parsons 

Introduction

Silver stain method

Procedure

  1. Fix 2-18 hrs. (50% MeOH/12% HoAc, 0.05% formalin), 1 liter/gel
  2. Wash three times in 35% EtOH/water for 20 min
  3. Wash twice in distilled water for 10 minutes
  4. Sensitize 2 min with 0.5L of 100 mM sodium thiosulfate, 30 mM potassium ferricyanide
  5. Wash in 0.5 L water for 10 min. four times
  6. Stain 20 min. with 1 L 0.2% silver nitrate, 0.076% formalin
  7. Wash in water 1 min. twice
  8. Develop till dark enough with 1 L of 6% sodium carbonate, 0.05% formalin, 0.0004% sodium thiosulfate
  9. Stop 5 min. with 1 L of 50% MeOH/12% HoAC
  10. Store in 1% HoAc/water Desiderio Silver Destain
  11. Water 5 min (3X)
  12. Destain whole gel 15 min. with 1:1 v/v 30mM potassium ferricyanide and 100 mM sodium thiosulfate prepared fresh
  13. Water 5 min. (10X)
  14. Proceed to Vorum procedure and fix for 2 hrs and redo silver stain

*Destain is useful if background darkens faster than the protein bands or if the gel is not stopped quickly enough and is generally too dark.

References

  1. Vorum and Blum procedures: presented at 48th American Society for Mass Spectrometry Conference on Mass Spectrometry June 11-15 2000, Long Beach, CA, poster TPE 191.
  2. Desiderio procedure: R.R. Becklin, E.S. Umstor, D.M. Desiderio, poster WPF 216, 48th American Society for Mass Spectrometry Conference on Mass Spectrometry June 11-15 2000, Long Beach, CA
  3. For an excellent comparison of visible staining procedures see: Lin JF, Chen QX, Tian HY, Gao X, Yu ML, Xu GJ, Zhao FK. Anal Bioanal Chem. 2008 Apr;390(7):1765-73.

Author information

T Parsons, Unaffiliated

Source: Protocol Exchange (2010) doi:10.1038/nprot.2009.218. Originally published online 5 January 2010.

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