Bio-Rad Silver Stains - Manual (Page 5)

Section 5
Protocols
Table 1. Silver Stain Protocol
Begin with step 1 immediately after the electrophoretic run is complete and fol-
low through to step 13 as indicated. For a convenient stopping point, gels can be
stored in 40% methanol/10% acetic acid (step 1 ) indefinitely.
Duration
<0.5 0.5-1.0 >1.0
mm mm mm
Reagent Volume* gel gel gel
1. Fixative 40% methanol/10% acetic 400 ml 30 min 30 min 60 min
acid (v/v)
2. Fixative 10% ethanol/5% acetic acid (v/v) 400 ml 15 min 15 min 30 min
3. Fixative 10% ethanol/5% acetic acid (v/v) 400 ml 15 min 15 min 30 min
4. Oxidizer 200 ml 3 min 5 min 10 min
5. Deionized water 400 ml 2 min 5 min 10 min
6. Deionized water 400 ml 2 min 5 min 10 min
7. Deionized water 400 ml — — 10 min
Repeat washes 5, 6, 7 until
all the yello color is
removed from the gel.
8. Silver reagent 200 ml 15 min 20 min 30 min
9. Deionized water 400 ml — 1 min 2 min
10. Developer 200 ml~30 sec. Develop until
solution turns yellow or
until brown “smokey”
precipitate appears.
Then pour off developer,
and add fresh developer.
11. Developer 200 ml ~5 min ~5 min ~5 min
12. Developer 200 ml — ~5 min ~5 min
13. Stop 5% acetic acid (v/v) 400 ml ~5 min ~5 min ~5 min
* Recommended volumes for a 16 cm slab gel in a standard 21 x 21 x 5 cm baking dish. For
smaller gels in a smaller container, volumes may be lowered proportionally. For example, for a
mini vertical gel (8 x 7 cm) you can decrease all volumes by one-half. The glass container should
be of appropriate size to allow free movement of the gel during shaking while maintaining a
volume of reagent sufficient to cover the gel completely.
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Section 4
Reagent Preparation
Prepare the oxidizer and silver reagent solutions on the same day that staining
is to be performed:
Full Size Gels Mini Gels
ml Reagent ml H
2
O Total ml ml Reagent ml H
2
O Total ml
Oxidizer 20 180 200 10 90 100
Silver Reagent 20 180 200 10 90 100
Developer Dissolve one bottle in 3.6 liters of deionized water by stirring
for 15 minutes at room temperature. This is enough for 6 full
sized gels or 12 mini gels. To prepare smaller amounts, use 32
grams of developer per liter of deionized water. Be sure to shake
the bottle thoroughly before weighing out, since the compo-
nents will settle out and separate. Failure to mix the contents
can result in very slow or no development. Store the solution at
23-25 ˚C for up to 1 month. Keep the solution tightly covered to
avoid evaporation of paraformaldehyde.
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