Bio-Rad Fluorescent Protein Stains User Manual

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)

................................................................................................................................

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

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.

2

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).

3

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)