Silver Staining of Acrylamide Gels

互联网2013-09-06

687

Inc. Time
1. Gel Fix Solution (50% MeOH, 12% TCA, 2% CuCl2)	20 min.
100% MeOH	250ml	<center> (Good for 1 month)</center>
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.

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