Bio-Rad Silver Stains - Manual (Page 6)

Section 6
Additional Applications
6.1 Nucleic acid staining
Follow the silver staining protocol, deleting the acetic acid from the fixatives
(steps 1, 2, and 3, Table 1). Double and single stranded DNA and RNA in poly-
acrylamide gels can be stained. Silver staining is not recommended for agarose gels.
6.2 Silver staining after Coomassie Brilliant Blue R-250
staining
Completely destain the Coomassie stained gel in 40% methanol/10% acetic acid.
Begin at step 2 of the silver staining protocol and proceed as usual.
6.3 Silver staining electrofocusing slab gels
Fix the gel in 30% methanol/10% trichloroacetic acid/3.5% sulfosalicylic acid
for 1 hour. Follow by at least 2 hours in several volumes of 30% methanol/12%
trichloroacetic acid to insure complete removal of ampholytes. Go to step 2 of the
protocol and proceed as indicated. Store stained gels in 40-50% methanol (other
low % gels may also be stored this way to control swelling). Modifications for gels
bonded to a polyester backing: If gel is < 0.5 mm thick, modify wash steps 5
through 7 to 2 x 5 minutes in 400 ml deionized water. Increase step 9 to 1 minute
in 400 ml deionized water. For gels > 0.5 mm thick, modify steps 5 through 7 to
2 x 10 minutes and increase step 9 to 1 minute in 400 ml deionized water.
Section 7
Troubleshooting Guide
Problem Solution
Gray or brown precipitate Non-specific deposition of silver due to
appearing as smudges or swirling oxidizer or silver reagent carry-over. Increase
on gel surface. May become wash steps:
mirror-like. Bands may be faint
or absent.
gels
≤
1 mm: Increase steps 5, 6, and
7(including any omitted steps) to 5 min-
utes each. Increase step 9 to 2 minutes.
gels >1 mm: Increase steps 5, 6, and 7 to
20 minutes each.
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Table 2. Modified Silver Stain Protocol
This modified protocol, optimized for mini gels (~7 cm x 8 cm x 0.75 mm), gives
clear backgrounds and consistent results in less time than the standard protocol.
Incubation times may need to be increased for large format gels.
Reagent Duration
1. Fixative: 40% methanol/10% 30 minutes minimum. Gel may be
acetic acid (v/v) stored overnight at this step.
2. Oxidizer: 5 minutes
Note: make sure gel is completely
immersed.
3. Water Washes: use large volumes 15 minutes maximum
of water and change the wash many
times (6-7), especially in the first 5
minutes. This flushes the oxidizer
from the gel without removing it
from the proteins. Proceed to step
4 after 15 minutes even if gel is still
slightly yellow in color.
4. Silver Reagent: 20 minutes
5. Quick Water Rinse: 30 seconds maximum
6. Developer: ~ 30 seconds or until a brown or
smokey precipitate appears. Quickly
pour off the solution and add fresh
developer. Repeat this step if precipi-
tate appears again. If the solution
remains clear, the gel can remain in
developer for about 5 minutes.
Change developer every 5 minutes
until desired intensity is obtained.
7. Stop: 5% acetic acid (v/v) 15 minutes
Note: make sure gel is completely
immersed.
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