Silver Staining of Acrylamide Gels

Inc. Time

20 min.

250ml

60ml

10g

to 500ml

 10 min.

 100ml

 50ml

 850ml

 1000ml

 10 min.

  0.02g

 200ml

 200ml

  10 min.

  5 min.

  5 min.

 10 min.

  0.2g

 200ml

200ml

  20 sec.

  1 min.

  until developed

 40ml

 160ml

 54ul

 200ml

  1-2 min.

 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.