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Silver Staining of Acrylamide Gels

Silver Staining of Acrylamide Gels

From: W. Ansorge, J. of Biochem and Biophys. Methods, 11(1985):13-20

Inc. Time
1. Gel Fix Solution (50% MeOH, 12% TCA, 2% CuCl2)

20 min.

100% MeOH

250ml

(Good for 1 month)
100% TCA

60ml

 
CuCl2

10g

 
H2O

to 500ml

 
 2. Solution A (10% EtOH, 5% Acetic Acid)

 10 min.

 100% EtOH

 100ml

 
 100% Acetic Acid

 50ml

 
 H20

 850ml

 
 

 1000ml

 
 3. 0.01% KMnO4

 10 min.

 KMnO4

  0.02g

 
 dH2O

 200ml

 
 

 200ml

 
 4. Solution A (from Above)

  10 min.

 5. 10% EtOH

  5 min.

 6. dH2O

  5 min.

 7. 0.1% AgNO3

 10 min.

 AgNO3

  0.2g

 
 dH2O

 200ml

 
 

200ml

 
 8. dH2O dip

  20 sec.

 9. 10%K2CO3

  1 min.

 10. Developer (2% K2CO3, 0.01% Formaldehyde)

  until developed

 10% K2CO3

 40ml

 
 dH2O

 160ml

 
 37% Formaldehyde

 54ul

 
 

 200ml

 
 11. Solution A (from above)

  1-2 min.

 12. H2O

 15 min

   

Comments:

  
  • All gel treatments should use around 200 mls solution with slow agitation.
  • Gels may first be stained (and destained) with coomassie blue before silver staining.
  • For increased sensitivity: After development in step 10 and 10 min. H2O wash, one may repeat the procedure from step 7.
  • Times of step 1 and 5 are important, esp. for thicker gels (>0.2mm).
  • When gels cannot be stained immediately after electrophoresis:
  • 1. fix gel 15 min. in 50%MeOH, 20% TCA.
  • 2. store gel in 10% EtOH, 5% Acetic Acid
  • 3. When ready, begin with step 1 and proceed normally
  • KMnO4 and AgNO3 solutions should be prepared fresh, and AgNO3 should be stored in the dark until use.
  • Dark background could be due to use of old gel solutions. (esp. BIS)
  • Remove stacker before beginning staining procedure.
  • Stain will fade slightly if left in water for several hours following staining procedure. However, once dried the stain looks very pronounced again.

 

Contributed By: Dave Walter

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Last Update: 12/03/1998