Lonza Rockland, Inc.
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Tech Service: 800-521-0390
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Document # 18168-1007-02
Rockland, ME 04841 USASYPRO® Ruby Protein Gel Stain
Introduction®

Tips for Staining

SYPRO Ruby Protein Gel Stain is a highly sensitive
fluorescent stain for the rapid detection of proteins inpolyacrylamide gels. This stain provides excellent sensitivity
for standard 1-D SDS-PAGE, 2-D PAGE, and isoelectric
focusing (IEF). The detection sensitivity exceeds Coomassie®
Brilliant Blue Stain and provides comparable sensitivity tothat of the best silver staining techniques (1ng-2 ng per®
band). SYPRO Ruby Stain has the following advantages oversilver staining:•Simple staining protocol•
Less protein to protein variability•
Stains a wide variety of difficult–to-stain proteins
including lipoproteins, calcium binding proteins,glycoproteins and fibrillar proteins•
No possibility of overstaining•
Does not interfere with mass spectrometry or Edmanbased sequencing•
Provides a linear quantitation range of over threeorders of magnitude••••••General Procedure for Staining Proteins withSYPRO® Protein Gel Stains®

SYPRO Ruby Stain can be used with a variety of gels
including Tris-Glycine, Tris-Tricine, and nondenaturing.

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Contents®

SYPRO Ruby Stain is available in three sizes and supplied
ready to use. The three sizes provide enough material to stain
the following number of gels:•••

Provide continuous, gentle agitation – An orbital shaker
step at ~ 50 rmp will produce the proper level of agitation.
Stain in polypropylene dishes – The high-density plasticof these containers adsorb a minimal amount of dye andincrease staining efficiency. Rubbermaid® Servin’®
Savers are an example of an appropriate container.
For large format gels, stain in polyvinyl chloride
photographic staining trays – 8x10 photographic trays
work well.
Containers should be clean – Rinse containers withethanol prior to use.
Use circular staining dishes for staining small gels –The use of these containers results in less dye
aggregation and better staining.
Glass dishes are not recommended.

Run gels according to standard protocols.
Fix the gel (NOTE: fixation is not required for 1-D SDSpolyacrylamide gels). Any of the following solutions canbe used to fix 2-D gels. Fix for 20 minutes:•••••

200 ml - ~ 4 minigels
1 L - ~ 20 minigels or 2-3 large format gels
5 L - ~ 100 minigels or 10-15 large format gels

10% methanol/7% acetic acid
25% ethanol/12.5% trichloroacetic acid10% ethanol/7% acetic acid50% ethanol/3% acetic acid40% ethanol/10% acetic acid

NOTE: Combining ethanol and acetic acid can result in theformation of the toxic compound ethyl acetate which may
interfere with identification of proteins via mass spectroscopy).

Storage and Handling®

SYPRO Ruby Stain should be stored at room temperatureand protected from light. Under these conditions, the stain isstable for at least 9 months.

Fix IEF gels in a 40% methanol/10% trichloroacetic acidsolution for three hours followed by three, ten minute washes
in dH2O.

When using the 5 L size, store the box on its side with top flap
closed in order to protect the stain from light.

Disposal
Stain solutions should be disposed of by passing throughactivated charcoal followed by incineration of the charcoal.
All federal, state, and local environmental regulations shouldbe observed when disposing of the stain.

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Place the gel into the staining container and cover with alid to protect from light. The container can also bewrapped in aluminum foil to further shield the stain fromlight during the staining process. The following are theminimum volumes that should be used to stain a gel of agiven size:•
50 ml/8 cm x 10 cm x 0.75 mm gel•
330 ml/16 cm x 20 cm x 1 mm gel•
500 ml/20 cm x 20 cm x 1 mm gel•
Approximately 10 times the volume of a given gel
is needed for efficient staining.

••

acrylamide side down on the transilluminator and use anemission filter to screen out the blue fluorescence of theplastic.
Laser scanning instruments that emit at 450 nm, 473 nm,488, or 532 nm can be used to visualize gels stained with®
SYPRO Ruby Stain.

Identification of Individual protein spots
®

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Since SYPRO Ruby Stain does not bind covalently toproteins, it can be used in conjunction with Edman-based
sequencing or mass spectroscopy after staining with nosubsequent interference from the stain.

Gently agitate the gel at room temperature.
Stain 1-D and 2-D gels for a minimum of three hours.
Stain IEF gels overnight.
•The optimal staining time depends on thethickness of the gel and the gel concentration.
Longer staining times may be required as gel
thickness increases.
•Increasing the staining time can increase
sensitivity.
•Gels will not overstain.
•Diluting the stain from the concentration at which
it is supplied will decrease sensitivity.
•Reuse of the staining solution will result in asignificant loss of sensitivity.
Transfer the gel to a clean staining dish and wash with a
10% methanol (or ethanol)/7% acetic acid solution for
30 minutes. This will minimize background fluorescence.
IEF gels require this procedure to be repeated three times
in order to achieve the best signal to background ratio.
Gels can be monitored using UV epi-illumnination todetermine the level of background fluorescence.
Remove the gel from the staining container andphotograph the gel following the procedures outlined
below.
The gel can be dried for permanent storage; however,proteins present at very low levels may no longer be
detectable after drying. Incubate the gel in a 2% glycerol
solution for 30 minutes prior to drying it using a gel dryer.

Photographing Gels Stained with SYPRO® ProteinGel Stains
SYPRO® Ruby Stain has two excitation maxima (at ~280 nm
and ~450 nm), and one emission maximum (at ~610 nm). The
stained gels can be visualized using a 300 nm UVtransilluminator, a blue light transilluminator, or a laser®
scanner. SYPRO Ruby Stain remains photostable over longexposure times.
••••

Clean the surface of the transilluminator prior to and after
each use using deionized water and a soft cloth.
Fluorescent dyes can accumulate on the glass surface
and cause unacceptable background fluorescence.
The highest sensitivity using a Polaroid® camera is®
achieved using Polaroid 667 black-and-white print film
and 490 nm longpass filter. Set the f-stop at 4.5 andexpose for 1 second.
The highest sensitivity using a CCD camera is achieved
using 1024 x 1024 pixels resolution with 12- or 16-bit grayscale levels per pixel. Contact the camera’s manufacturer
NOTE: These parameters can vary depending upon thefor recommendations on which filter sets to use type oftransilluminator being used.
The backing on some precast gels is highly fluorescent.
When using gels with a backing such as this, place the gel

2SYPRO® Stain Troubleshooting Guide

Symptom
No Bands seen on gel

CausesSolution/Explanation

Wrong 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 transferbuffer®
No SYPRO stained
bands visible onWestern blot aftertransfer
Large blob of stain atbottom of gel
Proteins not visible onnative gels
Protein not visible on
Phast™ system gels orgels backed withGelBond® PAG Film®

Photobleaching of SYPRO
StainsWrong methanol concentration

Proteins with low hydrophobicity®

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

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 and Orange’s
staining and may quench 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 canresult 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 excitation and emissionmaxima).
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 during®
transfer, 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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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 at www.Lonza.com
GelBond is a trademark of FMC Corp. SYPRO is a trademark ofMolecular 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 Chemicals Industries,
PLC. All other trademarks herein are marks of the Lonza Group or its
affiliates.

© Lonza Rockland, Inc.
All Rights reserved.

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