Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18112-0807-04
Rockland, ME 04841 USA

SYPRO® Red & Tangerine Protein Gel Stains

Introduction

Disposal

SYPRO® Protein Gel Stains are highly sensitive fluorescent
stains for the rapid detection of proteins in polyacrylamide gels.
The detection sensitivity exceeds Coomassie® brilliant blue,
approaches that of silver stain and can detect as little as 1 ng
– 8 ng of protein per band. Simply soak the gel in a solution of
1X SYPRO® Stain and see results within 40 – 60 minutes
without destaining. These stains do not require a separate
fixation step. Gels stained with SYPRO® Protein Gel Stains
exhibit low background and minimal protein-to-protein staining
variability. Gels can be documented using standard imaging
systems.

Stain solutions should be disposed of by passing throughactivated charcoal followed by incineration of the charcoal.

Tips for Staining••

Staining gel before
Western transfer

Tangerine

Zymography••

Green He-Ne or
Nd:YAG lasers

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Red

Argon laser-based gel
scanners

Mass Spectrometry

SYPRO® STAIN

Routine highperformance staining

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Contents
500 μl of the 5,000X stock solution is enough to prepare 2.5
liters of working stain solution.

Storage and Handling••

SYPRO® Stains are supplied as 5,000X concentrated
stock solutions in DMSO. Store dessicated at room
temperature protected from light. When stored per
directions the stock solutions are stable for 6 to 12 months
from the date of receipt.
Diluted stock solutions are stable for at least three months
when protected from light and stored at 4°C in a clean,
detergent-free glass or plastic bottle.

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Cautions
The DMSO stock solutions should be handled with caution as
DMSO is known to facilitate the entry of organic molecules into
tissues. We strongly recommend using double gloves
when handling the DMSO stock solutions. Please refer tothe MSDS for more details. No data are available on the
toxicity of the SYPRO® Protein Gel Stains.

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For gels to be transferred, use SYPRO® Tangerine
Protein Gel Stain.
Before you stain - Run SDS-polyacrylamide gelsaccording to standard protocols. Prepare and run gelsusing 0.05% SDS. Gels with 0.1% SDS may require aslightly longer staining time or a 10 minute rinse in waterto reduce the background.
Do not fix the proteins in the gel using methanolcontaining solutions. Methanol removes the SDS coatfrom the proteins, strongly reducing the detection signal
from the stains.
Staining the gel in transfer buffer will result in
decreased sensitivity. For blotting techniques, werecommend staining the gel with SYPRO® Tangerine
Protein Gel Stain which does not require an acetic acidfixation step. Dilute SYPRO® Tangerine Gel Stain in 50
mM phosphate, 150 mM NaCl, pH 7.0.
The SDS front at the bottom of the gel stains heavily with
SYPRO® Red and Tangerine Stains. Unless the protein ofinterest co-migrates with the SDS front we recommend
running the SDS front off the end of the gel.
Colored stains such as Coomassie® blue stains andcolored protein markers may interfere with SYPRO®
staining and quench fluorescence. To stain gels previously
stained with Coomassie® blue stain, soak the gel inseveral changes of 7.5% acetic acid to remove the
Coomassie stain. Then incubate the gel in 0.05% SDS for
30 minutes and stain with SYPRO® Stain as usual.
Glove powder can leave background markings on gels.
Rinse or wash gloves prior to handling gels.
Clean the surface of the transilluminator after each use
with deionized water and a soft cloth. Fluorescent stains(such as SYPRO® Stains or ethidium bromide) can
accumulate on the transilluminator and may cause highbackground.
Handle gels carefully to avoid non-specific staining ofareas of the gel that have been squeezed.
SYPRO® Stains may be photobleached after several
minutes of exposure to UV light. If a gel becomes
photobleached, restain by incubating in the staining
solution.

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SYPRO® stained gels can be restained with Coomassie®
blue or silver stain procedures.
Plastic wraps and GelBond® Film will autofluoresce when
exposed to UV light resulting in very high background. For
imaging backed gels, we recommend the use of Clare
Chemical’s Dark Reader® transilluminator.

If proteins are to be used for subsequent analysis dilute
SYPRO® Tangerine stain stock solution into 50 mM
phosphate, 150 mM NaCl, pH 7.0 or use one of the buffers
listed above prepared as 50 mM – 100 mM solutions
containing 150 mM NaCl.

General Procedure for Staining Proteins with
SYPRO® Protein Gel Stains

Staining Proteins for Subsequent Analysis such as
Zymography or Western Blotting

1.

Stained proteins can also be eluted from gels and used forfurther analysis such as mass spectrometry.
NOTE: If the gel will be blotted after staining with SYPRO®
Tangerine Stain, stain the gel according to the procedure
below, using an appropriate diluent (previously described). Add
0.1% SDS to the Transfer Buffer. This may help in the transfer
of some proteins to the membrane.
1. Run SDS-polyacrylamide gels according to standard
protocols.
NOTE: It is not necessary to decrease the amount of SDS
present in the running buffer when using SYPRO®
Tangerine Gel Stain.
2. Dilute the 5,000X concentrate to a 1X solution of diluent ina clear plastic polypropylene container. For example, forevery 10 ml diluent add 2 µl stock SYPRO® Stain solution.
3. Mix well.
4. Place the gel into the staining container and cover toprotect from light.
5. Gently agitate the gel at room temperature.
6. Stain the gel for 40-60 minutes.
NOTE: The optimal staining time depends on the
thickness of the gel and the gel concentration. Longer
staining times may be required as gel thickness increases.
7. Remove the gel from the staining container and
immediately photograph the gel following the procedures
outlined below.

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3.
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8.

Run SDS-polyacrylamide gels according to standard
protocols.
NOTE: To reduce background staining with SYPRO® Red
Stain use 0.05% SDS in the running buffer. Gels run in
0.05% SDS show no change in the migration pattern ofproteins.
Dilute the 5,000X concentrate to a 1X solution, in 7.5%(v/v) acetic acid, in a clear plastic polypropylene container.
For example, for every 10 ml acetic acid add 2 µl stock
SYPRO® Stain solution.
Mix well.
Place the gel into the staining container and cover with alid to protect from light.
Gently agitate the gel at room temperature.
Stain the gel for 40-60 minutes.
NOTE: The optimal staining time depends on the
thickness of the gel and the gel concentration. Longer
staining times may be required as gel thickness increases.
Briefly rinse the gel in 7.5% acetic acid.
Remove the gel from the staining container andphotograph the gel following the procedures outlined
below.

Staining Nondenaturing Gels with SYPRO® Red
Protein Gel Stain
There are 2 options for staining proteins with SYPRO® Red
Stain after native gel electrophoresis.
Option 1
Dissolve the stain 1:5,000 in distilled water and follow thegeneral protocol for staining proteins with SYPRO® Protein
Gel Stains.
NOTE: This will be highly protein-selective, and will be lesssensitive than staining proteins in SDS gels. To increase thesignal, a long film exposure can be used since the background
fluorescence is essentially zero.
Option 2
Soak the gel after electrophoresis in 0.05% SDS for 30
minutes, then stain with a 1:5,000 solution of SYPRO® Stain
diluted in 7.5% acetic acid.
NOTE: Proteins will be denatured and fixed after
electrophoresis using this treatment.

Viewing Gels Stained with SYPRO® Protein Gel
StainsAll SYPRO® Protein Gel Stains have two excitation
wavelengths; in the UV region at approximately 300 nm andthe visible region between 470 and 610 nm. This makes it
possible to visualize the stains using a UV transilluminator, theDark Reader® transilluminator from Clare Chemical, CCD
camera systems or laser scanners.
SYPRO® Stain
Red
Tangerine

Staining Proteins with SYPRO® Tangerine Protein
Gel Stain: Non-fixing Protocol
SYPRO® Tangerine Protein Gel Stain is an extremely versatilefluorescent stain that does not alter protein structure orinterfere with the transfer of proteins to blotting membranes.
The stain can be diluted into a wide range of buffers with a pH
range from 4-10 and if fixing is necessary, it can be diluted into
7.5% acetic acid following the General Protocol for Staining
Proteins with SYPRO® Gel Stains. SYPRO® Tangerine
Protein Gel Stain is compatible with the following buffers:
Formate pH 4.0
Citrate pH 4.5
Acetate pH 5.0
MES pH 6.0
Imidazole pH 7.0

HEPES pH 7.5
Tris acetate pH 8.0
Tris-HCl pH 8.5
Tris borate, 20 mM EDTA pH 9.0
Bicarbonate, pH 10.0

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Emission
630
640

Excitation (nm)
550, 300
490, 300

Photographing Gels Stained with SYPRO® Protein
Gel Stains
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Restaining Procedures
Gels stained with SYPRO® Protein Gel Stains can berestained with either Coomassie® brilliant blue or with silver
stain. Follow your standard protocol.

Protein bands stained with SYPRO® Protein Gel Stains
are best seen by photographing the gel. The integrating
effect of a camera or imaging system can detect bands
that are not visible to the eye.
Photobleaching can occur after extended (several
minutes) exposure to UV light. If bleached, the gel can berestained with only a small decrease in sensitivity.
We recommend SYPRO® Red Stain for green HE-Ne or
Nd:YAG laser.
The highest sensitivity using a Polaroid® camera can beobtained using Polaroid 667 black-and–white print film andthe SYPRO® Protein Gel Stain Photographic Filter.
Exposure time will vary with the intensity of the illumination
source. Begin with an f-stop of 4.5 and an exposure of
2 - 8 seconds. Use of an ethidium bromide filter is notrecommended as it blocks much of the light and leads tolower detection sensitivity.
The SYPRO® Photographic Filter does not work with CCD
camera systems. For CCD cameras use the emission andexcitation data above and check with the camera
manufacturer for the appropriate filter.

Gels stained with Coomassie® brilliant blue can be restainedwith SYPRO® Stains. To stain gels previously stained with
Coomassie® brilliant blue stain, you must remove the
Coomassie® brilliant blue as it will quench the SYPRO®
Stains. Soak the gel in either 30% methanol or 7.5% acetic
acid with several changes of the destaining solution. Incubate
the gel in 0.05% SDS for 30 minutes. Stain with SYPRO®
Stain following the general protocol.

Staining Gels for Western Blot Transfer
SYPRO® Stains can be diluted in Western blot transfer buffer
to stain gels that will subsequently be transferred. The stains
do not interfere with antibody probing of proteins on themembrane or microsequencing as they are removed from theproteins during the Western blot procedure. Staining in transfer
buffer is about 2- to 4-fold less sensitive versus staining inacetic acid. Proteins are not commonly visible on membrane
after transfer. For staining when optimal protein activity isdesired, use SYPRO® Tangerine Protein Gel Stain.
For SYPRO® Red Gel Stain, use a transfer buffer containing
25 mM Tris, 187 mM glycine and 20% methanol. Higher
methanol concentrations, or SDS in the transfer buffer will
result in reduced protein detection. For SYPRO® Tangerine
Gel Stain dilute the stock solution in 50 mM phosphate, 150
mM NaCl, pH 7.0.
1.
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3.
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6.

Briefly centrifuge the vial containing the stain to deposit
the solution at the bottom of the vial.
Dilute an appropriate volume of the 5,000X stock,(2 μl/10 ml) in the appropriate diluent (previously
described).
Mix vigorously.
Stain the gel for 40 to 60 minutes as described in thegeneral staining protocol.
Follow the instructions for “Photography”.
Transfer the proteins to a membrane using standard
protocols and fresh transfer buffer.

Destaining Procedures
Overnight incubation in 0.1% Tween® 20 will remove most of
the stain. Soaking in several changes of 7.5% acetic acid willremove all of the stain. Soaking in methanol will strip off thedye, the SDS, and will also precipitate the proteins.

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SYPRO® Stain Troubleshooting Guide
Symptom
No Bands seen on gel

CausesWrong UV light box
Very little protein
Incorrect stain dilution
Use of colored stains, marker
dyes or prestained protein
markers
Stain container

Old running buffer

Destaining the gel
No bands inphotograph

High background.
High background

Wrong exposure
Improper photographic filters

Bands visible on lightbox, but fade with time
No bands visible afterstaining in transfer
bufferNo SYPRO® stained
bands visible onWestern blot after
transfer
Large blob of stain atbottom of gel
Proteins not visible onnative gels
Protein not visible on
Phast™ System gelsor gels backed with
GelBond® PAG Film

Photobleaching of SYPRO®
StainsWrong methanol concentration

Proteins with low hydrophobicity

Binding of SYPRO® Stains to
SDS front.
SYPRO® Stains require
SDS/protein complexes to bindto proteins
SYPRO® Stain binding to
GelBond® Film, which strongly
autofluoresces

Solution/Explanation
Use a UV light box with 300 nm-312 nm lamps. Be sure the lamps
are producing appropriate intensity.
At the lower end of SYPRO® Stain’s sensitivity range, the protein
bands may not be visible by eye, but will show in the photograph.
Diluting the stain below the recommended concentration will result
in reduced staining sensitivity.
Colored stains or marker dyes, as well as commercially prestained
protein markers, may interfere with SYPRO® Red Stain andquench fluorescence. Use unstained markers.
Clean and rinse the staining dishes well before use as thedetergent will interfere with staining. We recommend pipette-tip lid
boxes, Rubbermaid® Servin’Saver® or Seal-A-Meal® type bags.
SDS can precipitate out of the running buffer, decreasing thestain’s ability to bind. Use either fresh running buffer, or add SDS
concentrate to buffer just prior to use.
SYPRO® Stains do not require destaining. If required refer todestaining procedures.
Use the correct stain dilution. A higher staining concentration can
result in increased background and quench fluorescence.
The high background can sometimes obscure the protein bands.
Use of the proper filters during photography will decrease
background, allowing the bands to be visible. Decreasing the gel's
SDS concentration to 0.05% from 0.1% may also help reduce
background.
Vary exposure until the background is low and the protein bands
are visible. See protocol for recommended exposures.
Use the recommended photographic filters. For systems that use
Wratten® Filters, either the SYPRO® Filter or a Wratten® #9
works best. Use a Tiffen® #15 (yellow-15) Filter for systems that
require threaded glass filters. For CCD cameras, consult the
manufacturer for the appropriate filter (see protocol for excitationand emission maxima).
Restain the gel in fresh stain in the appropriate staining buffer.
Use recommended methanol concentration in transfer buffer/stain.
High methanol concentrations can strip SDS from the proteins,
resulting in low detection levels.
Only highly hydrophobic proteins will retain enough SYPRO® Stain
to be visible on a membrane. SDS is stripped off proteins duringtransfer, resulting in very little retention of the SYPRO® Stain on
most proteins.
Run gel longer, or if running small proteins, change to Tris-Tricine
buffer system.
Refer to procedure for staining nondenaturing proteins.

Remove GelBond® Film from the gel prior to staining.
Use Clare Chemical’s Dark Reader® transilluminator to image
gels.

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Ordering Information
Catalog No. Description

Quantity

50542
50543
50556

10 x 50 μl
500 μl500 µl50540

SYPRO® Red Stain
SYPRO® Red Stain
SYPRO® Tangerine Stain
SYPRO® Photographic
FilterEach

Related Products
PAGEr® Precast Gels
ProSieve® Protein Marker
ProSieve® 50 Gel solution

For Research Use Only.
Manufactured for Lonza.
Product licensed from Molecular Probes, Inc.
For more information contact Technical Service at(800) 521-0390 or visit our website atwww.Lonza.com

GelBond is a trademark of FMC Corp. SYPRO is a trademark of
Molecular Probes, Inc. Wratten is a trademark of Eastman Kodak Co.
Tiffen is a trademark of Tiffen Manufacturing Corp. Dark Reader is atrademark of Clare Chemical. Tween is a trademark of ICI Americas,
Inc. Rubbermaid and Servin’ Saver are trademarks of Rubbermaid,Inc. Seal-A-Meal is a trademark of Dazey Corp. Phast is a trademarkof Pharmacia LKB Biotechnology, Polaroid is a trademark of Polaroid
Corp. Coomassie is a trademark of Imperial Chemical Industries. Allother trademarks herein are marks of the Lonza Group or its affiliates.

© 2007 Lonza Rockland, Inc.
All rights reserved.

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