SYPRO Orange and SYPRO Red Protein Gel Stains

SYPRO Orange and SYPRO Red Protein Gel Stains
Product Information
Revised: 17-January-2003
SYPRO® Orange and SYPRO® Red Protein Gel Stains
Storage upon receipt:
• Storage temperature not critical
• Desiccate
• Protect from light
• SYPRO Orange dye: 300, 470/570 nm
• SYPRO Red dye: 300, 550/630 nm
Molecular Probes’ proprietary SYPRO® Orange and
SYPRO® Red protein gel stains provide fast, simple, sensitive
staining of proteins in electrophoretic gels and offer the following
advantages over conventional colorimetric stains:
$ High sensitivity. SYPRO Orange and SYPRO Red protein
gel stains can detect 4–8 ng of protein per minigel band,
higher sensitivity than Coomassie® brilliant blue (CBB) staining and as sensitive as many silver staining techniques (Figure 1).
$ Rapid staining. Staining is completed in less than an hour.1,2
$ Simple staining procedure. After electrophoresis, the gel is
simply stained, rinsed and photographed — no separate fixation or destaining steps are required, and there is no fear of
overstaining the gel.1,3
$ Compatibility with standard laboratory equipment.
Stained proteins can be visualized using a standard 300 nm
UV transilluminator or a laser scanner (Figure 2).
$ Low protein-to-protein variability. Because SYPRO
Orange and SYPRO Red dyes interact with the SDS coat
around proteins in the gel, they give more consistent staining
between different types of proteins compared to CBB staining1 and never exhibit negative staining. SYPRO Orange and
SYPRO Red stain glycoproteins well.
$ High selectivity for proteins. SYPRO Orange and SYPRO
Red protein gel stains detect a variety of proteins down to
~6500 daltons without staining nucleic acid or lipopolysaccharide contaminants that are sometimes found in protein
preparations derived from cell or tissue extracts.
$ Broad linear range of detection. The fluorescence intensity
of SYPRO dye–stained bands is linear with protein quantity
over three orders of magnitude, a much broader range than
either CBB or silver staining can provide.1
MP 06650
Figure 1. Identical polyacrylamide minigels stained with A) SYPRO
Orange gel stain, B) SYPRO Red gel stain, C) silver stain and
D) Coomassie brilliant blue (CBB) stain according to standard protocols. The SYPRO dye–stained gels were photographed using 300 nm
transillumination, a SYPRO photographic filter (S-6656)and Polaroid
667 black-and-white print film. The CBB- and silver-stained gels were
photographed using transmitted white light and Polaroid 667 blackand-white print film; no optical filter was used.
The staining properties of SYPRO Orange and SYPRO Red
dyes are similar, and both are equally suitable for use in most
procedures. The SYPRO Orange gel stain is slightly brighter,
whereas the SYPRO Red gel stain has somewhat lower background fluorescence. Both dyes are efficiently excited by UV or
broadband illumination (Figure 2) and, with the proper filters,
work well with CCD camera archiving systems. For those using
a laser scanner, choose the dye with the excitation maximum
most closely matching the excitation light in the scanner (Figure
SYPRO Orange and SYPRO Red protein gel stains are not
suitable for staining proteins on blotting membranes and they
show greatly reduced sensitivity when staining proteins in IEF or
2-D gels. For these applications, we recommend our SYPRO
Ruby protein blot stain (S-11791), and SYPRO Ruby protein gel
stain (S-12000, S-12001, S-21900), respectively.
SYPRO Orange protein gel stain (S-6650, S-6651) and
SYPRO Red protein gel stain (S-6653, S-6654) are provided as
SYPRO® Orange and SYPRO® Red Protein Gel Stains
5000X concentrated solutions in dimethylsulfoxide (DMSO),
either as a single vial containing 500 µL of stock solution or as
a set of 10 vials, each containing 50 µL of stock solution. In
each case, enough reagent is supplied to prepare a total of 2.5 L
of working stain solution, which is sufficient to stain ~50 polyacrylamide minigels.
of dye are present, they should be redissolved by briefly sonicating the tube or vortexing it vigorously after warming.
We must caution that no data are available addressing the
toxicity of the SYPRO protein gel stains. The DMSO stock
solution should be handled with particular caution as DMSO is
known to facilitate the entry of organic molecules into tissues.
We strongly recommend using double gloves when handling
DMSO stock solutions. Solutions of SYPRO stains should be
poured through activated charcoal before disposal. The charcoal
must then be incinerated to destroy the dye. We have found that
1 g of activated charcoal binds at least 98% of the SYPRO
Orange or SYPRO Red dye present in 2.5 L of 1X staining solution prepared in 7.5% acetic acid, which is equivalent to the
amount of dye in 500 µL of the 5000X concentrated DMSO
We also offer the SYPRO Protein Gel Stain Starter Kit
(S-12012), which includes:
SYPRO Orange protein gel stain, 50 µL
SYPRO Red protein gel stain, 50 µL
SYPRO Tangerine protein gel stain (S-12010), 50 µL
SYPRO protein gel stain photographic filter (S-6656)
The SYPRO stock solutions should be stored protected from
light at room temperature, 4°C or –20°C. When stored properly,
these stock solutions are stable for six months to one year. The
staining reagent diluted in buffer or acetic acid solution can be
stored in very clean and detergent-free glass or plastic bottles,
protected from light at 4°C for at least three months.
SDS-Polyacrylamide Gel Electrophoresis
Prepare and run SDS-polyacrylamide gel according to standard protocols.4,5 For the highest sensitivity, we recommend
using 0.05% SDS in the running buffer (instead of the usual
0.1% SDS). Gels run in 0.05% SDS show no change in the
migration of proteins and can be photographed more quickly
because they require less time in the staining solution to clear the
SDS from the gel. Gels run in SDS concentrations lower than
0.05% or in old running buffer exhibit poor resolution of bands
and other problems, so it is essential that the SDS stock solution
used to prepare the running buffer be fresh and at the proper SDS
Before opening, each vial should be allowed to warm to
room temperature and then briefly centrifuged in a microfuge to
deposit the DMSO solution at the bottom of the vial. If particles
Staining Proteins in the Gel
Staining Proteins After Electrophoresis
1. Prepare the staining solution by diluting the stock SYPRO
reagent 1:5000 in 7.5% (v/v) acetic acid and mixing
$ Diluting the stain below the recommended concentration will
result in reduced staining sensitivity.
$ Using higher staining concentrations than recommended will
not result in better detection, but will instead result in increased background in the gel and quenching of the fluorescence from dye molecules crowded around the proteins.
$ The staining solution may be reused up to four times. However, we see dramatically reduced sensitivity, especially after
the second reuse and therefore recommend using fresh staining solution for optimal sensitivity.
$ For low percentage gels and for very small proteins, 10% acetic acid solution will result in better retention of the protein in
the gel without compromising sensitivity.
$ Acetic acid will interfere with transfer of the proteins to a
blot. For Western blotting and other blotting techniques, you
may dilute the SYPRO Orange or SYPRO Red stain in standard transfer buffer. However, staining the gel in transfer
buffer will result in lower sensitivity staining. Therefore, for
blotting techniques, we recommend staining the gel with
SYPRO Tangerine protein gel stain (S-12010), which does
not require acetic acid fixation, or staining the blot directly
with SYPRO Ruby protein blot stain (S-11791).
Figure 2. The fluorescence excitation and emission spectra of
A) SYPRO Orange and B) SYPRO Red protein gel stains diluted
1:10,000 in water containing 0.05% SDS and 150 µg/mL bovine serum
SYPRO® Orange and SYPRO® Red Protein Gel Stains
teins in SDS gels; however, because there is essentially no background fluorescence, photographic exposures can be very long.
If it is not necessary to maintain the protein in a native state
after electrophoresis, the best sensitivity can be achieved if the
gel is soaked in 0.05% SDS for about 30 minutes and then
stained with a solution of the SYPRO dye diluted in 7.5% acetic
$ Do not fix the proteins in the gel with methanol-containing
solutions. Methanol removes the SDS coat from proteins,
strongly reducing the signal from SYPRO Orange or SYPRO
Red protein gel stains.
2. Pour the staining solution into a small plastic dish.
$ For one or two standard-size minigels, use about 50 mL of
staining solution. For larger gels, use between 500 and
750 mL of staining solution.
$ We use Rubbermaid® Servin’ Saver containers with lids, but
also find that the lids of pipet boxes work just fine.
$ Clean and rinse the staining dishes well before use as detergent will interfere with staining.
$ Gels may also be stained in Seal-A-Meal® type bags. It is
still important to use the proper amount of staining solution.
Staining During Electrophoresis
SYPRO Orange and SYPRO Red protein gel stains can be
dissolved in the cathode (top) running buffer to stain proteins as
the gel runs. The SYPRO stock solution can be diluted 5000fold into the cathode running buffer. The dye moves through the
gel with the SDS front, so that all sizes of protein are stained.
Staining does not influence relative migration of proteins through
the gel. This method results in poorer protein staining than the
standard post-staining method and requires the same amount of
time as the gel must be destained for 15 to 40 minutes in 7.5%
acetic acid to reduce background fluorescence.
The SYPRO Red and SYPRO Orange stains cannot be used
to prestain protein samples for SDS gels. Loading solutions contain so much SDS that the dye simply localizes in the free SDS
and binds very little to the proteins. For nondenaturing gels it
might be possible to use SYPRO dyes as prestains, but we have
not tried this. SYPRO Red stain has been used as a pre-stain for
capillary electrophoresis.6
3. Place the gel into the staining solution.
$ Cover the container with aluminum foil to protect the dye
from bright light.
4. Gently agitate the gel at room temperature.
$ The staining time is 10 to 60 minutes, depending on the
thickness and percentage of the gel. For 1 mm–thick 15%
poly-acrylamide gels, the signal is typically optimal at 40 to
60 minutes of staining.
$ Once the optimal signal is achieved, additional staining time
(several hours to overnight) does not enhance or degrade the
signal. Gels can be left in stain for up to a week with only a
small loss in sensitivity; our detection limits under these conditions are approximately 8–16 ng/band.
Viewing and Photographing the Gel
Gels may be left in staining solution overnight without losing
sensitivity. However, the fixation in acetic acid is relatively mild,
so for low percentage gels or very small proteins, photographs
should be taken as soon as possible after staining, before the proteins begin to diffuse.
5. Rinse briefly with 7.5% acetic acid.
$ This brief rinse (less than a minute) removes excess stain
from the gel surface to reduce background fluorescence on the
surface of the transilluminator or gel scanner.
$ Molecular Dynamics has stated that 30 minutes of destaining
in 7.5% acetic acid improves background and signal detection
in their gel scanner. However, our own testing has shown
that, for Polaroid 667® black-and-white photography, even a
10 minute destaining results in lower sensitivity.
Viewing the Gel
Gels may be visualized on a standard 300 nm UV transilluminator or with a blue-light transilluminator like the Clare Chemical Dark Reader™. We recommend cleaning the surface of the
transilluminator with water and a soft cloth after using to minimize the buildup of fluorescent dyes on the surface.
$ Place the gel directly on the transilluminator. Plastic wraps,
such as Saran® Wrap, fluoresce on their own and even more
when exposed to SYPRO Orange or SYPRO Red stain. This
gives a large background signal if the gel is sitting on a piece
of plastic wrap on a UV transilluminator and makes it impossible to get good sensitivity.
$ Pharmacia PhastGels® have polyester backing material
(Gelbond®) which is not only highly autofluorescent, but also
binds the SYPRO stains, producing additional background
fluorescence. Consequently, the plastic backing should be
removed before trying to visulaize your results. Pharmacia
markets a gel backing remover for use with their Phast
Transfer™ system.
$ For those using a laser-excited gel scanner, we recommend
the SYPRO Orange stain for argon laser–based instruments
and the SYPRO Red stain for instruments that employ green
He-Ne or Nd:YAG lasers (Figure 2).
Triton X-100 Gels
Triton® X-100 at 0.1% or greater will interfere with SYPRO
dye staining. If Triton X-100 is used with your gel, we recommend soaking the gel in two to three changes of buffer to be sure
the Triton X-100 is diluted out, and then incubating the gel in
0.05% SDS for 30 minutes before staining as usual.
2-D Gels and IEF Gels
SYPRO Orange and SYPRO Red protein gel stains are not
suitable for staining proteins in IEF gels, and they show reduced
sensitivity when staining proteins on 2-D gels. For these applications, we recommend SYPRO Ruby protein gel stain
(S-12000, S-12001, S-21900).
Nondenaturing Gels
Protein can be stained after native gel electrophoresis by dissolving the SYPRO dyes in water and then following the protocol above.
Staining proteins in nondenaturing gels is highly proteinselective and will generally be less sensitive than staining pro-
SYPRO® Orange and SYPRO® Red Protein Gel Stains
Photography with a Polaroid Camera
Destaining the Gel
Photographing the gel is essential to obtain high sensitivity.
The camera’s integrating effect can make bands visible that are
not visible to the eye. The highest sensitivity with a Polaroid®
camera will be obtained using Polaroid 667 black-and-white
print film and the SYPRO photographic filter (S-6656).7
Gels may be mostly destained by incubation overnight in
0.1% Tween® 20. Alternatively, incubation in several changes
of 7.5% acetic acid will eventually remove all of the stain. Incubation in methanol will strip off dye and SDS but will also precipitate proteins.
$ Standard ethidium bromide filters should not be used as they
will block much of the light and lead to lower sensitivity.
Supplemental UV blocking filters are not usually required.
$ Polaroid 667 film is a fast film with an ISO rating of ASA
3000. The use of different film types may require longer
exposure times or different filters.
$ Exposure time will vary with the intensity of the illumination
source: with an f-stop of 4.5, typically 2 to 5 seconds for
SYPRO Orange stain and 3 to 8 seconds for SYPRO Red
$ We generally observe detection limits of ~50 ng protein/band
with 300 nm transillumination and ~4–8 ng protein/band in a
photograph taken with Polaroid 667 black-and-white print
film. Our detection limits of 4–8 ng/band are obtained using
a Fotodyne® Foto/UV® 450 Ultraviolet Transilluminator,
which has six 15-watt bulbs that provide peak illumination at
312 nm. When using weaker illumination sources, exposures
must be adjusted accordingly.
$ Noticeable photobleaching can occur after several minutes of
exposure to ultraviolet light. If a gel becomes photobleached,
it can be restained by simply returning it to the staining solution.
$ The SDS front at the bottom of the gel stains very heavily
with SYPRO Orange and SYPRO Red stains. Unless the
proteins of interest are co-migrating with the SDS front, it
will be advantageous to run the SDS front off the gel.
$ Colored stains and marker dyes, as well as commercially prestained protein markers, interefere with SYPRO dye staining
and quench fluorescence.
$ Highly-colored prosthetic groups (e.g. heme) that remain
bound in native gels will quench fluorescence of the SYPRO
Orange and SYPRO Red stains.
$ Odd marks on stained gels can be caused by several factors.
If the gel is squeezed, a mark appears that stains heavily with
the SYPRO dyes. This is probably a localized high concentration of SDS that has difficulty diffusing out. Glove powder
can also give background markings, so we recommend rinsing or washing gloves prior to handling gels.
$ Staining with the SYPRO Orange dye occasionally results in
gels with scattered fluorescent speckles. We don’t know what
the speckles are and have not been able to completely get rid
of them, but they seem to be only a cosmetic problem — they
don’t reduce the dye’s sensitivity.
$ SYPRO dye–stained gels can be restained with either
Coomassie brilliant blue or with silver stain procedures.
In fact, for some silver staining methods, we have found that
prestaining with SYPRO dyes actually increases the rate of
staining and the sensitivity for detection.
$ To stain gels previously stained with Coomassie brilliant blue
stain, the stain must be completely removed as it will quench
the fluorescence of SYPRO dyes. Soaking the gel in either
30% methanol or 7.5% acetic acid with several changes of the
destaining solution will be effective at removing the
Coomassie stain. Once the Coomassie dye has been removed,
the gel should be incubated in 0.05% SDS for 30 minutes
before staining with the SYPRO stain as usual.
Photography with a CCD Camera
CCD cameras also provide good sensitivity, however the
SYPRO photographic filter may not be optimal. Contact the
manufacturer of your camera system for the optimal filter sets
to use.
Storing the Stained Gel
Gels may be stored by keeping them protected from light in
the staining solution. Although the signal does decrease somewhat after several days, your gels may retain a usable signal for
many weeks, depending on the amount of protein in your bands.
Gels may be dried between sheets of cellophane, although
there is sometimes a slight decrease in sensitivity. Store the
dried gel in the dark to prevent photobleaching.
$ If the gels are dried onto paper, the light will scatter and the
sensitivity will decrease.
$ If the gel is dried between sheets of other plastic, the plastic
typically used is not transparent to UV light.
1. Anal Biochem 239, 223 (1996); 2. J NIH Res 7, 82 (1995); 3. Anal Biochem 239, 238 (1996); 4. Short Protocols in Molecular Biology, Second Edition,
Ausubel et al, John Wiley & Sons (1992); 5. Nature 227, 680 (1970); 6. Electrophoresis 19, 2169 (1998); 7. Anal Biochem 248, 168 (1997).
SYPRO® Orange and SYPRO® Red Protein Gel Stains
Product List
Current prices may be obtained from our Web site or from our Customer Service Department.
Cat #
Product Name
Unit Size
500 µL
SYPRO® Orange protein gel stain *5000X concentrate in DMSO* ...........................................................................................
SYPRO® Orange protein gel stain *5000X concentrate in DMSO* *special packaging* .......................................................... 10x50 µL
SYPRO® photographic filter .......................................................................................................................................................
1 kit
SYPRO® Protein Gel Stain Starter Kit .......................................................................................................................................
SYPRO® Red protein gel stain *5000X concentrate in DMSO* ................................................................................................
500 µL
SYPRO® Red protein gel stain *5000X concentrate in DMSO* *special packaging* ............................................................... 10x50 µL
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SYPRO® Orange and SYPRO® Red Protein Gel Stains
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