Lonza Rockland, Inc.
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Document # 18167-1007-03
Rockland, ME 04841 USASYPRO® Ruby Protein Blot StainIntroduction
SYPRO® Ruby Protein Blot Stain is a highly sensitive
fluorescent stain for the rapid detection of proteins following
transfer to common blotting membranes such as nitrocellulose
and polyvinylidene difluoride (PVDF). Using SYPRO® Ruby
Blot Stain prior to performing specific protein detection
techniques provides an assessment of protein transferefficiency, allows for the detection of contaminating proteins,and enables the comparison of proteins of interest to molecularweight standards. SYPRO® Ruby Protein Blot Stain provides
the following:•Simple staining protocol, completed within 1 hour•
Specific for proteins. Will not detect contaminating
nucleic acids•
Fully compatible with fluorogenic, chemiluminescent,and colorimetric detection techniques•
No possibility of overstaining•
Does not interfere with mass spectrometry or Edmanbased sequencing•
High sensitivity•Contents

6.

SYPRO® Ruby Protein Blot Stain is available in a 200 ml bottle
and supplied ready-to-use. This is sufficient volume forstaining 10-40 minigel electroblots or four large format
electroblots (~20 x 20 cm). This stain can be reused up to fourtimes without significant loss in sensitivity.

Staining Proteins Blotted onto PVDF Membranes•

Staining Proteins blotted onto Nitrocellulose
Membranes
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1.

Storage and Handling
SYPRO® Ruby Protein Blot Stain should be stored at room
temperature and protected from light. Under these conditions,the stain is stable for 2 years.

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

Tips for Staining••••

Provide continuous, gentle agitation for all washingand staining steps – An orbital shaker step at ~ 50 rpm
will produce the proper level of agitation.
Stain in polypropylene dishes – The high-density plasticof these containers adsorb a minimal amount of dye and®
increase staining efficiency. Rubbermaid® Servin’ Savers
are an example of an appropriate container.
For large format gels, stain in polyvinyl chloride
photographic staining trays – 8 in x 10 in photographic
trays work well.
Containers should be clean – Rinse containers withethanol prior to use.

Use circular staining dishes for staining small
membranes – The use of these containers results in lessdye aggregation and better staining.
Glass dishes are not recommended.

2.

3.
4.
5.

6.

Run gels and electroblot according to standard protocols.
Fix the membrane in a solution of 7% acetic acid/10%
methanol at room temperature for 15 minutes. Ensurethat the membrane is fully immersed during fixation.
Wash membranes in four consecutive changes ofdeionized water for 5 minutes each.
®
Stain using SYPRO Ruby Protein Blot Stain for 15
minutes. Ensure that the membrane is fully immersed in
the staining solution.
Wash the membrane in five consecutive changes ofdeionized water for 1 minute each. Greater or fewer
washes can be done to maximize sensitivity and minimize
background fluorescence.
Membranes can be dried for permanent storage. After
staining, do not touch the membranes as this can destroy
the staining pattern. Use forceps to handle the wet blots.
Dry membranes can be handled freely.

Run gels and electroblot according to standard protocols.
After electoblotting proteins onto a PVDF membrane,
allow the membrane to dry completely prior to proceeding.
Fix the membrane in a solution of 7% acetic acid/10%
methanol at room temperature for 15 minutes. Float themembrane face down to ensure fixation.
Float the membrane in four changes of deionized water for
five minutes each to wash.
Float the membrane in SYPRO® Ruby Protein Blot Stain
for 15 minutes.
Wash the membrane 3 times in deionized water for 1
minute each to remove excess dye and reduce
background fluorescence.
Membranes can be dried for permanent storage. Afterstaining, do not touch the membranes as this can destroy
the staining pattern. Use forceps to handle the wet blots.
Dry membranes can be handled freely.

Photographing Membranes Stained with SYPRO®
Ruby Protein Blot StainSYPRO® Ruby Protein Blot Stain has two excitation maxima(at 280 nm and 450 nm), and one emission maximum(at 618 nm). The stained gels can be visualized using a 300nm UV transilluminator, a blue light transilluminator, or a laser®
scanner. SYPRO Ruby Protein Blot Stain remains
photostabile over long exposure 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 front face of membranes can be illuminated by either
using a handheld UV-B (~300 nm) light source, or byplacing a UV light box on its side to illuminate the blots.
Alternatively, a top illuminating system such as theBio-Rad Fluor-S™ can be used.
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
for recommendations on which filter sets to use.
NOTE: These parameters can vary depending upon thetype of transilluminator being used.
Laser scanning instruments that emit at 450 nm, 473 nm,488 nm, or 532 nm can be used to visualize gels stained®
with SYPRO Ruby Protein Blot Stain.

Post Blotting Analysis
Since SYPRO® Ruby Protein Blot Stain does not bindcovalently to proteins, immunostaining, glycoprotein staining ormass spectrometry can be performed immediately afterstaining. Since over 90% of SYPRO® Ruby Protein Blot Stain
is washed off the blot during the blocking step ofimmunostaining, it is important to document the staining prior
to beginning the immunostaing procedure. If performing
Edman-based sequencing subsequent to blotting, use a PVDF®
membrane, stain with SYPRO Ruby Protein Blot Stain as
above and then perform the following destain procedure:
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2.
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Place the membrane face down in a solution of 150 mM
Tris (pH 8.8)/20% methanol for 10 minutes using gentle
agitation
Rinse the blot four times for 1 minute each in dH2O
Air dry the membraneSYPRO® Stain Troubleshooting GuideSymptom
No Bands seen on gel

CausesWrong UV light box
Very little protein
Incorrect stain dilution
Use of colored stains, marker
dyes or prestained proteinmarkers
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 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 toGelBond 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 staining 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 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 useWratten® 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 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® Stainto 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.

For more information contact Technical Service at(800) 521-0390 or visit our website atwww.Lonza.com

For Research Use Only. Not for Use in a Diagnostic
Procedure.
Manufactured for Lonza.
Product licensed from Molecular Probes, Inc.
GelBond is a trademark of FMC Corporation. SYPRO is atrademark of Molecular Probes, Inc. Wratten is a trademark ofEastman Kodak Co. Tiffen is a trademark of Tiffen
Manufacturing Corp. Dark Reader is a trademark of Clare
Chemical Research, Inc. Rubbermaid and Servin’ Saver aretrademarks of Rubbermaid Corporation. Seal-A-Meal is atrademark of Dazey Corp. Phast is a trademark of Pharmacia
LKB Biotechnology, Polaroid is a trademark of Polaroid Corp.
Fluor-S is a trademark of Bio-Rad Laboratories, Inc. All othertrademarks are marks of the Lonza Group or its affiliates.
©2007 Lonza Rockland, Inc.
All rights reserved.