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Flamingo
™
Fluorescent
Gel Stain
Instruction Manual
Catalog Numbers
161-0490, 20 ml
161-0491, 100 ml
161-0492, 500 ml
For technical support call your local Bio-Rad office, or in the US call
1-800-4BIORAD (1-800-424-6723)
................................................................................................................................
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Table of Contents
Section 1 Introduction and General Information........................1
1.1 Introduction.........................................................................1
1.2 Product Description ............................................................1
1.3 Storage...............................................................................1
1.4 Materials and Equipment Required but not Supplied ..........2
1.5 Reagents Required but not Supplied ..................................2
1.6 Safety Considerations .........................................................2
1.7 Disposal Considerations......................................................2
1.8 Fluorescence Characteristics ..............................................2
Section 2 Instructions .....................................................................4
2.1 General Considerations.......................................................4
2.2 Prepare Fix Solution............................................................4
2.3 Fix Gels...............................................................................4
2.4 Prepare Stain Solution ........................................................5
2.5 Stain Gels ...........................................................................5
2.6 Background Reduction (Optional) .......................................5
2.7 Gel Imaging.........................................................................5
Section 3 Technical Information....................................................6
3.1 Sensitivity with Laser Scanning ...........................................6
3.2 Sensitivity with UV Transillumination ....................................7
3.3 Protein-to-Protein Variability ................................................7
3.4 Linearity of Staining.............................................................8
Section 4 Troubleshooting ........................................................
Section 5 Product Information.......................................................9
4.1 Flamingo Fluorescent Gel Stain...........................................9
4.2 Related Products ................................................................9
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1
Section 1
Introduction and General Information
1.1 Introduction
Flamingo fluorescent gel stain allows sensitive fluorescence visualization of
proteins that have been separated by 1-D or 2-D SDS-PAGE. It comes as a 10x
stock solution that is diluted with water by the user to its working concentration. It
is supplied in three product sizes: 20 ml, 100 ml, and 500 ml.
Flamingo fluorescent gel stain contains a dye which binds to denatured protein.
This dye is normally non-fluorescent in solution, but becomes strongly fluorescent
when protein-bound. The staining procedure is a simple two-step protocol that
can be completed in as little as 5 hours. Gels to be stained are fixed with
ethanol/acetic acid solution prior to staining with the gel stain solution. A
destaining step is not normally required, but may be employed to reduce background.
Gels stained with Flamingo fluorescent gel stain may be visualized with a variety of
different fluorescence imaging systems. The most optimal imaging systems are
laser-based fluorescence scanners capable of exciting and detecting at 510 nm
and 535 nm respectively, such as the Molecular Imager FX
™
system. Gels may
also be imaged using systems that use 300 nm UV transillumination such as the
VersaDoc
™
imaging system.
Flamingo fluorescent gel stain gives exceptional sensitivity and dynamic range and
is compatible with subsequent analysis by enzymatic digestion and mass
spectrometry. It is thus particularly well-suited to proteomics applications. Stained
gels may be stored in the dark at 2–8°C for up to six months without significant
loss of imaging sensitivity.
1.2 Product Description
Flamingo fluorescent gel stain is a 10× stock solution that comes in three package
sizes. Instructions are included.
• The 20 ml size stains 4 mini gels (Ready Gel
®
) or 2 Criterion™gels.
• The 100 ml size stains 20 mini gels (Ready Gel), 10 Criterion gels, 4 PROTEAN
®
II gels, or 2 PROTEAN®Plus gels.
• The 500 ml size stains 100 mini gels (Ready Gel), 50 Criterion gels, 20 PROTEAN II
gels, or 10 PROTEAN Plus gels.
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1.3 Storage
The product is stable for at least 6 months when stored at 2–8°C. Avoid exposure
to temperatures greater than 37°C and protect from light.
1.4 Materials and Equipment Required but not Supplied
• Staining containers—Glass trays are recommended, plastic trays may be used if
cleaned and rinsed thoroughly
• Imaging equipment—Gels are best imaged using a laser-based fluorescence
scanner capable of exciting and detecting near 510 nm and 540 nm respectively,
such as the Molecular Imager FX system. Gels may also be imaged using
systems based on the UV transilluminator and CCD camera such as the
VersaDoc imaging system
• Laboratory shaker or rocker
• Powder-free latex, vinyl or nitrile gloves
1.5 Reagents Required but not Supplied
• Acetic Acid, reagent grade
• Ethanol, reagent grade
• Filtered, distilled or deionized water.
1.6 Safety Considerations
Flamingo fluorescent gel stain is a dilute alcoholic solution of an organic dye. It is
flammable in its 10x concentrated form and should be handled in a manner that
prevents exposure to open flame or sparks. The diluted working solution is
minimally hazardous and non-flammable. However, the complete properties of the
dye component have not been investigated. Eye protection and gloves should be
worn and general laboratory safety precautions followed while handling both the
diluted and undiluted product.
1.7. Disposal Considerations
Laws governing the disposal of laboratory chemicals vary by region. Consult the
MSDS and check local laws for the proper disposal guidelines.
1.8. Fluorescence Characteristics
In the presence of protein, Flamingo fluorescent gel stain has a fluorescence
excitation maximum of 512 nm and a fluorescence emission maximum of 535 nm.
Flamingo fluorescent gel stain has a secondary excitation at 271 nm and can also
be excited with UV light (Fig. 1).
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Fig. 1. Excitation (A) and Emission (B) spectra for Flamingo fluorescent gel stain in the
presence of protein (denatured carbonic anhydrase).
Excitation Wavelength (nm)
Emission Wavelength (nm)
A
B
100
80
60
40
20
Fluorescence intensity %
0
250 300 350 400 450 500 550 600
Exci tati on w a vele ngth (nm)
100
80
60
40
20
Fluorescence intensity %
0
450 500 550 600 650
Emission wavelength (nm)
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Section 2
Instructions
2.1 General Considerations
Best results are obtained using clean technique. Any dust or dirt transferred to the
surface of the gel may appear in the fluorescence image as smudges or speckles.
Flamingo fluorescent gel stain is exceptionally sensitive. Contaminant proteins
such as keratin will appear in the gel image if care is not taken to minimize such
contamination.
All glassware used should be cleaned with laboratory glassware cleaner and
rinsed with distilled or deionized water. Use dust-free gloves and limit dust
exposure by keeping reagent vessels and gel trays covered as much as possible.
If gels are cast in the laboratory, the glass plates used should be thoroughly
cleaned with lint-free laboratory wipes.
Flamingo fluorescent gel stain may be exposed to typical room light for up to 8 hours
without any observable effect on the fluorescence intensity of the stained protein
bands or spots. However, if gels are ok as is left in stain solution for more extended
periods, photobleaching may occur. This can be prevented by covering the gel
trays with aluminum foil or an opaque lid.
Instructions given are for standard 1 mm thick SDS-PAGE gels. Thicker gels may
benefit from longer fix and stain times and larger volumes of solution. Native PAGE
gels may also be stained with this procedure.
2.2 Prepare Fix Solution
The fix solution consists of 40% (v/v) ethanol and 10% (v/v) acetic acid. Prepare
the solution with distilled or deionized water that has been filtered. The quantity of
fix solution required depends on the number of gels to be stained and the size of
the gel as indicated in the table below.
Gel Size
Volume of Fix Solution
per Gel
Ready Gel (8.6 cm × 6.8 cm) 100 ml
Criterion (13.3 cm × 8.7 cm)
200 ml
PROTEAN II (16 cm × 16 cm)
500 ml
PROTEAN Plus (25.6 cm × 23 cm)
1,000 ml
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2.3 Fix Gels
Remove the gel(s) from the gel cassette or plates. Place gel in a clean glass tray
with the volume of fix solution indicated above. Cover the tray, place on a rocker
or shaker and agitate gently. Fix for at least 2 hours.
Note: Gels may be left in fix solution for up to 24 hours. Shortened fix time or
insufficient fix solution may reduce sensitivity.
2.4 Prepare Working (1x) Stain Solution
Working (1x) stain solution is prepared by diluting one volume of Flamingo
fluorescent gel stain with 9 volumes of distilled or deionized water that has been
filtered. The quantity of stain solution required depends on the number of gels to
be stained and the size of the gel as indicated in the table below.
Working (1x) stain solution should be prepared within one day of its intended use.
2.5 Stain Gels
Carefully pour off the fix solution and add Flamingo fluorescent gel stain to the
staining tray. Cover the tray, place on a rocker or shaker and agitate gently. Stain
for at least 3 hours.
Gels may be left in the stain solution for extended periods. If the gel is to be left in
the stain solution for longer than 8 hours, cover the gel tray with aluminum foil or
other opaque material to limit light exposure.
Stained gels may be stored for up to six months and imaged without significant
loss of sensitivity. Gels held for long-term storage should be placed in sealable
plastic bags with 5–10 ml of stain solution and stored in the dark at 2–8°C.
Gel Size
Volume of Stain Solution
per Gel
Ready Gel (8.6 cm × 6.8 cm) 50 ml
Criterion (13.3 cm × 8.7 cm)
100 ml
PROTEAN II (16 cm × 16 cm)
250 ml
PROTEAN Plus (25.6 cm × 23 cm)
500 ml
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2.6 Background Reduction (Optional)
Note: The dye in Flamingo fluorescent gel stain has very low fluorescence when
not protein-bound. A background reduction or destaining step is normally not
required but the following step will slightly lower background staining and may
allow some proteins to be more sensitively detected.
Carefully pour off the stain solution and replace with an equal volume of 0.1% (w/v)
Tween 20. Cover the tray, place on a rocker or shaker and agitate gently for
10 minutes. Rinse gels with distilled or deionized water prior to imaging.
2.7 Gel Imaging
The primary fluorescence excitation maximum of Flamingo fluorescent gel stain is
at 512 nm. There is a minor excitation maximum at 271 nm. The fluorescence
emission maximum is at 535 nm (see Section 1.8 Fluorescence Characteristics).
Flamingo fluorescent gel stain therefore gives the most sensitive results when used
with visible laser light-based imaging systems equipped with 473, 488 or 532 nm
laser light sources. The Molecular Imager FX system is particularly well suited for
imaging gels stained with Flamingo fluorescent gel stain, and provides high-resolution images suitable for proteomics applications. If the accompanying software
does not have a have a preprogrammed imaging function for Flamingo fluorescent
gel stain, the image settings for SYPRO
®
Ruby stain may be used. If an alternative
laser-based scanner is used, choose imaging settings that match the fluorescence
characteristics of Flamingo fluorescent gel stain. Examples of compatible excitation
sources and emission filter combinations include the following:
• Green laser (532 nm) with 555 nm longpass emission filter
• Blue laser (473 nm) with 530 nm emission filter
• Blue laser (488 nm) with 560 nm longpass emission filter
Flamingo fluorescent gel stain may also be used with UV transilluminator-based
systems such as the VersaDoc imaging system. However, the limit of sensitivity
with UV transillumination will not be as low as that achievable with a visible laser
light-based imaging system.
If UV transillumination is used, make sure that the transilluminator has 300 nm
bulbs installed.
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Section 3
Technical Information
3.1. Sensitivity with Laser Scanning
When scanned on the Molecular Imager FX system, an SDS-PAGE gel stained
with Flamingo fluorescent gel stain should exhibit a limit of sensitivity in the range
of 0.25–0.5 ng (Fig. 2).
Fig. 2. Serial 2-fold dilutions of SDS-PAGE molecular weight standards, broad range, on a
Criterion Tris-HCl 4–20% gel. The gel was stained with Flamingo fluorescent gel stain and imaged
with the Molecular Imager FX system. The arrow indicates the lane containing 0.5 ng of each standard.
0.5 ng
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3.2. Sensitivity with UV Transillumination
When scanned on the VersaDoc 4000 imaging system, an SDS-PAGE gel stained
with Flamingo fluorescent gel stain should exhibit a limit of sensitivity in the range
of 0.5–2 ng (see Fig. 3).
Fig. 3. Serial 2-fold dilutions of SDS-PAGE molecular weight standards, broad range, on a
Criterion Tris-HCl 4–20% gel. The gel was stained with Flamingo fluorescent gel stain and imaged
with the VersaDoc 4000 imaging system using an exposure time of 5 min. The arrow indicates the lane
containing 0.5 ng of each standard.
3.3. Protein-to-Protein Variability
Flamingo fluorescent gel stain will stain most proteins and exhibits less protein-toprotein variability than other staining methods.
3.4. Linearity of Staining
Flamingo fluorescent gel stain allows linear quantitation over a wide dynamic
range. Staining with the standard 5 hour protocol (2 hour fix, 3 hour stain) will
allow linear quantitation from 1 to 100 ng. If the staining step is extended to
overnight (at least 12 hours), the linear range for quantitation can be extended to
over three orders of magnitude (0.5 to 1 µg).
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A
2500
2000
1500
1000
fluorescence
500
0
012345
ng β-galactosidase
R2 = 0.9943
B
C
25000
20000
15000
10000
fluorescence
5000
0
0 10203040506070
300000
250000
200000
150000
100000
fluorescence
50000
0
0 200 400 600 800 1000 1200
ng β-galactosidase
ng β-galactosidase
R2 = 0.9947
R2 = 0.9960
Fig. 4. Serial 2-fold dilutions of ββ-galactosidase, run on a Criterion™ Tris-HCl 4–20% gel.
The gel was stained with Flamingo Fluorescent Gel Stain and imaged with the Molecular Imager FX
system. Fluorescence associated with the β-galactosidase band was determined using Quantity One
®
1-D Analysis Software. AA: Fluorescence associated with protein band in the range 0.25–4 ng. BB:
Fluorescence associated with protein band in the range 4–60 ng. CC: Fluorescnece associated with protein
band in the range 60–960 ng.
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Section 4
Troubleshooting
Problem Possible Cause Remedy
Bands or spots not No protein on gel Verify that there is actually
visible protein on the gel by
staining with another
method (e.g. Bio-Safe™
Coomassie stain).
Malfunctioning imaging Check instrument manual
system for troubleshooting.
Poor staining sensitivity Residual SDS in gel Confirm the correct amount
of fix solution is used. (See
page 5)
High or uneven Dirty equipment or Make sure that the staining
background staining staining trays trays and other equipment
used have been thoroughly
cleaned with laboratory
glassware cleaner. Use
glass staining trays, as
these are more likely to be
thoroughly clean.
Reagent impurities Make sure that the water
and reagents used for
preparing and fixing the gel
are of the highest possible
quality. Proteomics grade
water may be used to prepare the stain solution.
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Problem Possible Cause Remedy
Speckles or blotches in the Particulate material from Filter water used to prepare
gel image water, reagents, staining fix solution and stain
tray, ambient dust and solution if laboratory water
gloves system does not have a
filter.
Make sure that the staining
trays are thoroughly clean.
Limit the time that the gels
and staining solution are
exposed to open air.
Use dust-free gloves and
handle gels only by the
edges.
Particulate material may be
washed from the surface of
the gel prior to imaging by
washing with 0.1% (v/w)
Tween 20 prior to imaging
(See Section 2.6).
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Section 5
Product Information
4.1. Flamingo Fluorescent Gel Stain
Catalog # Description
161-0490 Flamingo Fluorescent Gel Stain, 10x Solution, 20 ml
161-0491 Flamingo Fluorescent Gel Stain, 10x Solution, 100 ml
161-0492 Flamingo Fluorescent Gel Stain, 10x Solution, 500 ml
4.2. Related Products
Catalog # Description
170-6531 Tween 20, EIA grade, 200 g
161-0781 10% Tween 20, for easy pipetting, 1 L
161-0786 Bio-Safe Coomassie Stain, 1 L
163-2097 Proteomics Grade Water, 500 ml
170-7856 Molecular Imager FX Pro Fluorescent Imager, PC
170-7857 Molecular Imager FX Pro Fluorescent Imager, Mac
170-7850 Molecular Imager FX Pro Plus MultiImager, PC
170-7851 Molecular Imager FX Pro Plus MultiImager, Mac
170-8140 VersaDoc Model 4000 Imaging System, PC
170-8141 VersaDoc Model 4000 Imaging System, Mac
170-8070 ChemiDoc XRS System, PC
170-8071 ChemiDoc XRS System, Mac
170-9600 Quantity One 1-D Analysis Software
170-9630 PDQuest 2-D Analysis Software
Tween is a trademark of ICE Americas Inc.
Coomassie is a tradmark of BASF Akfiengesellschaft.
Sypro is a registered trademark of Invitrogen.
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Life Science
Group
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