Bio-Rad Silver Stain
Catalog Numbers
161-0443
161-0444
161-0445
161-0447
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SIG 121393 Printed in USA
LIT34 Rev B
Bio-Rad
Laboratories
LIT34B 6/17/98 2:04 PM Page Cvr2
Table of Contents
Section 1 Introduction...............................................................1
Section 2 Silver Stain Kit Components ...................................1
Section 3 Important Procedural Information.........................1
3.1 Water Purity.........................................................................1
3.2 Gel Handling .......................................................................2
3.3 Temperature.........................................................................2
3.4 Convenient Stopping Point..................................................2
3.5 Band Development ..............................................................2
3.6 Destaining............................................................................2
3.7 Storage of Stained Gels .......................................................3
3.8 Photographing Gels .............................................................3
Section 4 Reagent Preparation.................................................4
Section 5 Protocols ....................................................................5
Section 6 Additional Applications............................................7
6.1 Nucleic Acid Staining..........................................................7
6.2 Silver Staining after Coomassie Blue R-250 Staining.........7
6.3 Silver Staining Electrofocusing Slab Gels...........................7
Section 7 Troubleshooting Guide.............................................7
Section 8 Ordering Information...............................................9
Section 9 References................................................................10
9.1 General ..............................................................................10
9.2 Proteins..............................................................................10
9.3 Nucleic Acids ....................................................................11
9.4 Isoelectric Focusing...........................................................11
LIT34B 6/17/98 2:04 PM Page i
Section 1
Introduction
Silver staining is a highly sensitive method for detecting proteins and nucleic
acids in polyacrylamide slab gels. The Bio-Rad Silver Stain, derived from the
method of Merril,1 is 10-50 fold more sensitive than Coomassie brilliant blue
R-250 for proteins (detection is ~0.1 ng/mm2) and 2-5 fold more sensitive than ethid-
ium bromide for single and double stranded DNA and RNA. Approximately 24 full
size gels or 48 mini gels can be stained with one kit.
Section 2
Silver Stain Kit Components
Oxidizer Concentrate - A 10-fold stock solution. Contains potassium dichro-
mate (CAS 7778-50-9) and nitric acid. Store at 4 ˚C.
Warning: Strong oxidizer - avoid contact with reducing agents.
Irritant - avoid contact with skin and eyes. Wear gloves and eye pro-
tection.
Silver Reagent Concentrate - a 10-fold stock solution. Contains silver nitrate
(CAS 7761-88-8). This solution is heat sensitive. Store at 4 ˚C.
Warning: Poisonous - caustic to eyes, skin, and mucous membranes. Wear
gloves and eye protection.
Developer - Four packages of dry chemical blend. Contains sodium carbonate and
paraformaldehyde (CAS 30525-89-4). Store at 4 ˚C.
Warning: Poisonous
Irritant - vapor and dust irritates eyes, mucous membranes, and skin.
Wear gloves, lab coat, and eye protection. Fume hood recommended.
Section 3
Important Procedural Information
Read the entire protocol, procedural information, and the Troubleshooting
Guide before beginning.
3.1 Water Purity
Deionized water of less than 1 µmho conductivity is recommended for all phas-
es of the procedure. Contaminants such as chloride ions will precipitate silver
ions causing reduced sensitivity and increased background. Water suspected of
containing interfering ions can be thoroughly deionized by passage over a column
of AG
®
501-X8(D) mixed bed ion exchange resin (catalog number 142-6452).
1
LIT34B 6/17/98 2:04 PM Page 1
fate pentahydrate in 1 liter deionized water. Prepare the destain by combining
equal parts of the two solutions immediately prior to use. Remove the gel from the
destain before complete destaining has occurred. Wash several times in deionized
water to remove the destain and prevent fogging. Stop the destaining process with
10% acetic acid for 15 minutes. Wash the gel for at least 1 hour with several
changes of water. To restain, begin with step 8, Table 1.
An alternative destain is Kodak rapid fix, solution A. Add 12 ml of rapid fix
to 88 ml deionized water to prepare the destain. Stop the destaining process with
2.4 g Kodak hypo clearing agent dissolved in 100 ml deionized water, for 15 min-
utes. Wash the gel for at least 1 hour with several changes of water. To restain,
begin with step 8, Table 1.
3.7 Storage of Stained Gels
After staining is complete, gels can be stored indefinitely in zip-lock plastic bags
with just a few drops of water. Alternatively, gels may be dried on filter paper.
Change the stop solution two or three times (at least 5 minutes for each step) to
remove all of the developer before drying. This will prevent continued development.
Drying gels between two pieces of cellophane also eliminates the problem of the
gel darkening upon drying.
3.8 Photographing Gels
Gels may be photographed during development or after stopping with 5%
acetic acid. Photographing should be done on a bright light box, however, devel-
opment may be accelerated if it has not yet been stopped. If the staining contain-
er is not optically clear, the gel should be placed on a glass plate or directly on the
light box for photographing.
Suggested Settings
Camera Polaroid-type 35 mm (50 mm lens)
(Polaroid Model MP-4)
Film Type 667, positive Kodak panatomic-X, asa 32
F stop F32 F16
Speed 1/125 sec. automatic setting
Height 1-2 feet 1-2 feet (on tripod)
3
3.2 Gel Handling
Wear rubber gloves that have been washed and rinsed with deionized water. Use
glass containers and make sure that the volumes are sufficient to allow free move-
ment of the gel during shaking. An orbital shaker is recommended to promote
mixing during all steps. Use a separate dish for each gel. Do not put pressure on
gels during handling or when decanting solutions.
3.3 Temperature
The oxidizer, silver reagent, and developer solutions should be at room tem-
perature (23-25 ˚C) for use. If heating is necessary, dilute the oxidizer and silver
reagent before warming to 25 ˚C. Use immediately. The developer can be heated
to 50 ˚C to enhance development (see “Band Development” below).
3.4 Convenient Stopping Point
Protein gels can be stored indefinitely in 40% methanol/10% acetic acid prior
to staining.
3.5 Band Development
Bands usually appear dark brown against a pale background. The duration of
the development steps is very approximate, and development should be monitored
closely. The third volume of developer may not be necessary, especially when a water
wash is used between the silver reagent and developer steps.
Stop development when the bands reach the desired intensity in relation to
background. If drying the gel on filter paper, stopping development just before
the desired intensity is reached will help keep the gel from turning darker on the
filter paper (see Troubleshooting Guide). The rate of development is highly tem-
perature dependent. Developer can be heated to 50 ˚C if faster development is
desired.
3.6 Destaining
Gels allowed to develop too long will have high background or surface deposits
of silver (mirroring). Destaining can be performed using a photographic reducer
such as the following, which was developed by Switzer, et al.
3
Dissolve 37 g of sodium chloride and 37 g of cupric sulfate anhydrous in 850
ml of deionized water. Add concentrated ammonium hydroxide until the precipi-
tate that forms is completely dissolved to give a deep blue solution. Adjust to 1 liter
with deionized water. Prepare a second solution containing 436 g sodium thiosul-
2
LIT34B 6/17/98 2:04 PM Page 2
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.
5
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.
4
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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.
7
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.
6
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Problem Solution
Some proteins are not staining. Silver stain as usual, then recycle gel.
Soak in deionized water for 30 minutes,
then repeat the silver staining starting with
the silver reagent step (step 8). Proteins
that did not stain on the first cycle will
stain to full intensity
Negative staining occurring. Proteins are overloaded. Recycle gel as
above. Some metalloproteins will always
stain negatively.
Silver reagent is being washed away.
Decrease time of wash in step 9.
Contaminant bands across Skin keratin (from dandruff, ungloved hands)
entire gel, occurring at 58 kD contaminating solutions. Wear gloves at all
and 64 kD. times during the pouring and running of
gels and solution preparation. Filter all
solutions through a 0.45 µm nitrocellulose.
Large discolored spots on gel. Any pressure on gel will cause the gel to
stain darker at that spot. Avoid crushing
the gel with fingers or dish when decant-
ing solutions or transferring gel to a new
solution.
Section 8
Ordering Information
Catalog
Number Product Description
161-0443 Silver Stain Kit, includes 1 bottle oxidizer concentrate, 1 bottle silver
reagent concentrate, and 4 bottles developer. Enough to stain approxi-
mately 24 16 cm slab gels.
161-0444 Oxidizer Concentrate, 480 ml
161-0445 Silver Reagent Concentrate, 480 ml
161-0447 Silver Stain Developer, 4 x 115 g
9
Troubleshooting Guide (continued)
Problem Solution
Temperature too low. Make sure temperature
of all silver stain reagents is at least 23 ˚C.
Mirroring can be caused by the developer
precipitate sticking to the gel surface. Be sure
to decant first development solution as soon
as the precipitate appears (see protocol).
Dark uniform background, usually Oxidizer is not completely removed.
Increase
yellow. wash steps 5, 6, and 7 as described above,
removing all traces of yellow, before
going on to silver reagent step.
Mottled background, usually Contaminants in water. Check that the
brown or green, with poor sensitivity. conductivity of the water is less than 1
µmho. See "Notes on Silver Staining."
Incomplete removal of gel buffer compo-
nents. Increase times of steps 1, 2, 3 to
insure complete removal.
Slow or no development. Rate of development is highly temperature
dependent. Developer solution can be
heated to 50 ˚C to speed development.
Developer solution is too old
(paraformaldehyde has evaporated). Shelf
life of solution is 1 month at 23-25 ˚C.
Settling and separation of the components
of the developer powder will occur. When
preparing solutions of less than 3.6 liters,
mix the contents thoroughly before
aliquoting.
Gel continues to develop or becomes Increase stop time (step 13) and change stop
darker upon drying onto filter paper. solution 2 or 3 times to remove all devel-
oper. Dry between two pieces of clear cel-
lophane or photograph before drying onto
filter paper.
8
LIT34B 6/17/98 2:04 PM Page 8
9.3 Nucleic Acids
12. Berry, M. J. and Samuel, C. E., “Detection of Subnanogram Amounts of RNA in
Polyacrylamide Gels in Presence and Absence of Protein by Staining with Silver,
Anal. Biochem., 124, 180 (1982).
13. McNeilage, L. J. and Whittingham, S., “Use of Bio-Rad Silver Stain to Identify Gel
Purified RNA Components of Small Nuclear Ribonucleoprotein Antigens,J.
Immunol. Methods, 66, 253 (1984).
14. Goldman, D. and Merril, C. R., “Silver Staining of DNA in Polyacrylamide Gels:
Linearity and Effect of Fragment Size,Electrophoresis, 3, 24 (1982).
15. Beidler, J. L., et al., “Ultrasensitive Staining of Nucleic Acids with Silver,Anal.
Biochem., 126, 374 ( 1982).
9.4 Isoelectric Focusing
16. Mehat, P. D. and Patrick, B. A., “Detection of Oligoclonal Bands in Unconcentrated
CSF: Isoelectric Focusing and Silver Staining,Neurology, 33, 1365 (1983).
17. Confavreux, C., et al., “Silver Stain After Isoelectric Focusing of Unconcentrated
Cerebrospinal Fluid: Visualization of Total Protein and Direct Immunofixation of
Immunoglobulin G,Electrophoresis, 3, 206 ( 1982)
11
Section 9
References
9.1 General
1. Merril, C. R., Goldman, D., Sedman, S. A. and Ebert, M. H., “Ultrasensitive Stain for
Proteins l
n
Polyacrylamide Gels Shows Regional Variation in Cerebrospinal Fluid
Proteins,Science, 211, 1437 (1981).
2. Laemmli, U. K., “Cleavage of Structural Proteins During the Assembly of the Head
of Bacteriophage T4", Nature, 227, 680 (1970).
3. Switzer, R. C., Merril, C. R. and Shifrin, S., “A Highly Sensitive Silver Stain for
Detecting Proteins and Peptides in Polyacrylamide Gels,Anal. Biochem., 98, 231
(1979).
4. O’Farrell, P. H., “High Resolution Two-Dimensional Electrophoresis of Proteins,J.
Biol. Chem., 250, 4007 (1975).
9.2 Proteins
5. Merril, C. R., Goldman, D., Sedman, S. A. and Ebert, M. H., “Ultrasensitive Stain for
Proteins in Polyacrylamide Gels Shows Regional Variation in Cerebrospinal Fluid
Proteins,Science, 211, 1437 (1981).
6. Merril, C. R. and Goldman, D., “Quantitative Two-Dimensional Protein
Electrophoresis for Studies of Iubom Errors of Metabolism,Clin. Chem., 28, No. 4
(1982), (reference on effect of recycling on image intensification).
7. Merril, C. R., Goldman, D. and Van Keuren, M. L., “Simplified Silver Protein
Detection and Image Enhancement Methods in Polyacrylamide Gels,
Electrophoresis, 3, 17 (1982).
8. Merril, C. R., Goldman, D. and Van Keuren, M. L., “Gel Protein Stains: Silver Stain,
Methods in Enzymology, 104, 441 (1984).
9. Merril, et al., “Molecular Probes for Human Genetic Diseases by Two-Dimensional
Electrophoresis and Silver Stain,” in: Electrophoresis '82, p. 327,Walter Degruyter,
publisher.
10. Merril, C. R. and Goldman, D., “Detection of Polypeptides in Two-Dimensional Gels
Using Silver Stain." in: Two-Dimensional Gel Electrophoresis of Proteins (Celis, J.
E. and Bravo, R., eds.), Academic Press, N.Y, 93 (1984).
11. Dzandu, L. K., Deh, M. E., Barratt, D. L. and Wise, G. E., “Detection of Erythrocyte
Membrane Proteins, Sialoglycoproteins and Lipids in the Same Polyacrylamide Gel
Using a Double-Staining Technique, Proc. Nat. Acad. Sci. USA, 81, 1733 (1984).
10
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