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

  • 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.