Staining Protocols

Staining Protocols
Application Bulletin
UVP AB-221
Internet: www.uvp.com
UVP, Inc. 2066 W. 11th Street, Upland, CA 91786
Tel: (800) 452-3197 (909)946-3197 Fax: (909) 946-3597 E-Mail: [email protected]
Ultra-Violet Products Ltd. Unit 1, Trinity Hall Farm Estate, Nuffield Rd., Cambridge CB4 1TG UK
Tel: +44(0)1223-420022 Fax: +44(0)1223-420022 E-Mail: [email protected]
STAINING PROTOCOLS
UVP has been a leading manufacturer of high
quality instrumentation for the detection of nucleic acids and proteins. Our wide selection of
transilluminators and epi-illuminators complements the ever expanding range of stains and
dyes on the market today. We offer transilluminators in various wavelengths, in the absorption spectra of popular fluorescent stains such
as ethidium bromide, SYBR Green, SYBR
Gold, SYPRO orange, and GelStar. The white
light transilluminator is useful when viewing protein dyes such as bromophenol blue and silver
stain. We have included short protocols of the
stains and dyes mentioned above for quick
reference. For more details please refer to the
web pages listed on the bottom of this bulletin.
destain it. View the gel with an UV transilluminator or hand-held lamps.
POST-STAINING WITH ETHIDIUM BROMIDE
Prepare enough ethidium bromide solution,
0.5µg to one ml of distilled water or running
buffer. Submerge the gel, after electrophoresis, in the solution for twenty minutes. Submerge the gel in distilled water for twenty minutes. Repeat with fresh distilled water. View
the gel with an UV transilluminator or hand-held
lamps.
SYBR GREEN I
EX/EM: 494/513
NUCLEIC ACID DETECTION IN
GELS
ETHIDIUM BROMIDE
EX/EM: 518/605
Ethidium bromide can be used to stain the gel
after electrophoresis or be incorporated into the
gel and buffer before electrophoresis. Higher
resolution can be obtained if the gel is stained
after running electrophoresis. Ethidium bromide is a mutagen and should be handled with
care.
PRE-STAINING WITH ETHIDIUM BROMIDE
Prepare agarose solution, add 0.5µg of
ethidium bromide to 1ml of the cooling solution.
Cast the gel. Add 0.5µg of ethidium bromide to
1ml of running buffer. Load and run the gel.
Submerge the gel in distilled water for twenty
minutes and again in fresh distilled water to
SYBR Green I is more sensitive then ethidium
bromide and less mutagenic. Like ethidium
bromide, SYBR Green I can be used after
electrophoresis or incorporated into the gel
before electrophoresis. Staining the gel after
electrophoresis results in higher resolution of
the bands. Incorporating the stain in the gel
can induce the gel to be more sensitive to DNA
overloading.
PRE-STAINING WITH SYBR GREEN I
Thaw the SYBR Green I solution. Spin solution
in a microcentrifuge. Add SYBR Green I to the
cooling agarose solution to a concentration of
1ml of SYBR Green I to 10000ml of gel solution. Cast the gel. Perform electrophoresis.
View the gel using a 300 nm UV transilluminator or 254 nm epi-illuminator. There is no need
for destaining.
Staining Protocols
2
POST-STAINING WITH GELSTAR
POST-STAINING WITH SYBR GREEN I
Thaw the SYBR Green I solution. Spin the
solution in a microcentrifuge. Dilute the stock
to 1X in a pH 7.0-8.5 buffer. Store in a clear
plastic polypropylene container.
Prepare
enough solution to just cover the top of the gel.
After running electrophoresis, place the gel in
the SYBR Green I solution at room temperature for 15-30 minutes depending on the thickness of the gel. View the gel using a 300 nm
transilluminator or 254 nm epi-illuminator.
There is no need for destaining.
SYBR GOLD
EX/EM: 495/537
SYBR Gold is used after electrophoresis, not
incorporated into the gel during electrophoresis.
Although there is no data pertaining to the
mutagenic properties of SYBR Gold, it should
be treated as a mutagen and be handled with
care.
STAINING WITH SYBR GOLD
Perform electrophoresis. Dilute stock SYBR
Gold to 1X with TBE, TAE, or TE, keeping a
pH of 7.0-8.0. Using a staining tray, submerge
the gel completely in solution. Keep the gel
and the solution out of the dark. Agitate the gel
at room temperature. Stain for 10-40 minutes,
depending on the thickness of the gel. View
with a 300nm UV transilluminator or 254nm
epi-illuminator.
GELSTAR
EX/EM: 493/527 (DNA)
493/532 (RNA)
GelStar can be used after electrophoresis or be
incorporated into the gel before electrophoresis. It is recommended to filter the gel buffer
prior to casting the gel since GelStar is sensitive to particles in the gel.
PRE-STAINING WITH GELSTAR
Thaw the GelStar solution and spin it in a microcentrifuge. Prepare gel solution. Add GelStar to the cooling gel solution. For DNA, add
stain solution so the final concentration is 1X.
For RNA, the final concentration should be 2X.
Cast the gel and run electrophoresis as practiced.
Perform electrophoresis. Thaw and spin the
GelStar stock solution. Prepare enough staining solution to completely submerge the gel.
Dilute stock in TAE, TBE, or TE buffer. For
DNA, the final concentration should be 1X, for
RNA, 2X. Place the gel and stain in a staining
tray. Agitate the gel while keeping the solution
away from light. The staining process is about
30 minutes, longer if the gel is thick.
SILVER STAIN
Silver staining in nucleic acids is as sensitive as
ethidium bromide. There are many silver staining kits available that yields higher sensitivity
than traditional staining. Silver staining can only
be used post electrophoresis.
POST-STAINING WITH SILVER STAIN
Reagents
1. Fixing solution: 500 ml methanol, 120 ml
glacial acetic acid, 50 g glycerol. Add distilled water to bring the volume to 1 l.
2. Staining solution:
a. Mix 50 g sodium carbonate and distilled
water to bring the volume to 1 l.
b. Mix 2.0 g ammonium nitrate, 2.0 g silver
nitrate, 10 g tunstosilic acid, 8 ml 37% formaldehyde. Add distilled water to bring the
volume to 1 l.
c. Mix 1:1 of the above two solution. Prepare
enough to completely submerge the gel.
3. Stop solution: Prepare 1% glacial acetic
acid in distilled water.
Protocol [1]
Submerge gel in fixing solution for thirty minutes. Wash the gel with distilled water for
twenty minutes. Using a staining tray, completely submerge gel in the staining solution
until the bands appear. Place the gel in the
stop solution for five minutes. Destain the gel
in distilled water and dry.
PROTEIN DETECTION IN GELS
COOMASSIE BRILLIANT BLUE
Coomassie blue non-specifically binds to proteins. It is faster and easier than silver staining.
It is not as sensitive a silver staining. Rapid
Coomassie blue staining can detect protein
bands after 5-10 minutes after the initial staining. The sensitivity is slightly reduced with rapid
staining.
Staining Protocols
STANDARD COOMASSIE BLUE STAINING
Reagents
1. Fixing solution: 50% (v/v) methanol, 10%
(v/v) acetic acid, 40% distilled water.
2. Staining solution: 0.05% (v/v) Coomassie
brilliant blue R-250, 50% (v/v) methanol,
10% (v/v) acetic acid, 40% distilled water.
Dissolve Coomassie brilliant blue in
methanol. Add acetic acid and water.
Store up to six months.
3. Destaining solution: 7% acetic acid, 5%
methanol, 88% distilled water.
Protocol [2]
Perform polyacrylamide electrophoresis. In a
plastic container, place gel and enough fixing
solution so the gel floats freely. Agitate for 2
hours on a rotary shaker. Pour out fixing solution, add staining solution to the container. Resume agitation for 4 hours. Pour out staining
solution. Rinse gel with 50 ml of fixing solution.
Pour out fixing solution and add destaining solution. Cover the gel in destaining solution and
agitate for 2 hours. Pour out destaining solution, add fresh destaining solution. Continue
the destaining process until clear blue bands
and background appear.
RAPID COOMASSIE BLUE STAINING
Reagents
1. Fixing solution: 25% (v/v) isopropanol, 10%
(v/v) acetic acid.
2. Rapid staining solution: 10% (v/v) acetic
acid, 0.0006 (w/v) Coomassie blue G-250.
Protocol
Perform polyacrylamide electrophoresis. In a
container, submerge the gel in the fixing solution. Shake gently at room temperature, 10-15
minutes for a 0.75-1.0 mm thick gel, 30-60
minutes for a 1.5 mm thick gel. Pour out fixing
solution, add rapid staining solution. Shake
gently for 2 hours to over night, until the desired
visibility of the bands is obtained. Pour out
staining solution. Cover the gel with 10% acetic acid to destain the gel. Shake gently for 2
hours. Repeat destaining process until a clear
background appears.
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It is more sensitive than Coomassie blue, detection limit at 2-5 ng/protein band. Silver staining is also more detrimental than Coomassie
blue. Gloves and fume hood should be used
while performing the staining process.
Reagents
1. Fixing solution: 50%(v/v) methanol, 10%
(v/v) acetic acid, 40% water
2. Destaining solution: 7% acetic acid, 16.5%
methanol, 78.5% water
3. Silver nitrate solution: add 3.5 ml concentrated NH4OH to 42 ml of 0.36% NaOH
and bring to 200ml with water. Mix, slowly
add 8 ml of 19.4% (16g/8ml) silver nitrate.
4. Developing solution: 0.5g sodium citrate,
0.5ml 37% formaldehyde solution, water to
100ml
5. 10% glutaraldehyde
6. Kodak Rapid Fix Solution A
Protocol [3]
Run polyacrylamide electrophoresis. Place the
gel and fixing solution in a staining container.
There should be enough solution to submerge
the gel. Agitate on an orbital shaker for 30 min.
Pour out fixing solution and pour in destaining
solution. Agitate on an orbital shaker for 60
min. Pour out destaining solution and pour in
10% glutaraldehyde and agitate under a fume
hood for 30 min. Pour out glutaraldehyde solution and wash 4 times with water, 30 min per
time. Pour out water, add enough silver nitrate
solution to submerge the gel. Vigorously shake
the gel for 15 min. Wash the gel 5 times with
deionized water, 1 min per time. Dilute 25 ml
of developing solution with 500 ml water.
Shake vigorously until the desired intensity of
the band appears. Change the solution if it
turns brown. Transfer the gel to Kodak Rapid
Fix Solution A for 5 min. Photograph gel.
SYPRO ORANGE
EX/EM: 470/560
SYPRO Orange is a fluorescent stain for protein analysis. It is much more sensitive than the
traditional silver and coomassie blue stains.
SYPRO Orange is also less time consuming,
10-60 minutes as opposed to overnight staining
required by certain gels.
STAINING WITH SYPRO ORANGE
SILVER STAIN
Silver staining is the binding of silver ions to the
sulfhydryl and carboxyl groups of the proteins.
Dilute stock SYPRO Orange solution o 1:5000
in 7.5% acetic acid. Pour the staining solution
in a small staining tray. Place the gel in the
staining tray and cover with aluminum foil.
Gently agitate the gel at room temperature for
Staining Protocols
10-60 minutes. The length of time is dependent on the thickness of the gel. Rinse briefly
with 7.5% acetic acid and view with a 300nm
transilluminator.
SYBR Green, SYBR Gold, and SYPRO Orange are
registered trademarks of Molecular Probes. For more
information
please
visit
their
website
at
http://www.probes.com
GelStar is a trademark of FMC BioProducts. For more
information
please
visit
their
website
at
http://www.bioproducts.com
REFERENCES
1. Your Complete Guide for DNA Separation and Analysis. “Detecting DNA with Silver Stain” FMC
BioProduct.
2. Ausubel, Frederick M. ed. Short Protocols in Molecular
Biology. “Coomassie Blue Staining”
John
Wiley & Sons: New York, 1992.
3. Short Protocols in Molecular Biology. “Silver Staining”
John Wiley & Sons: New York, 1992.
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