Fluorescent Protein Stains

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Overview

Specifications
Gel Stain
Shelf Life Storage Lowest Sensitivity Mass Spec Compatibility Recommended Imaging Systems
Flamingo 6 months 2–8°C 0.25–0.5 ng Yes Molecular Imager PharosFX
SYPRO Ruby 6 months Ambient 1–10 ng Yes Molecular Imager VersaDoc MP 4000
Oriole 12 months Ambient 1 ng Yes Molecular Imager GelDoc XR+
Imager Compatibility with Oriole Stain
Manufacturer Imaging System Recommended Settings
GE Healthcare ImageQuant UV 302 illumination with ethidium bromide (orange) emission filter
Fuji LAS 3000, LAS 4000 312 nm illumination with ethidium bromide emission filter
Alpha Innotech AlphaImager, FluorChem UV302 with standard (orange) emission filter
UVP BioDoc-It, VisiDoc-IT, DigiDoc-IT, MultiDoc-It, PhotoDoc-IT, BioSpectrum, EC3 Mid-range (302 nm) excitation with EtBr Red emission filter
Carestream Gel Logic UV trans excitation with 590 nm bandpass (ethidium bromide) emission filter
Kodak 2200, 4000MM, 4000MM Pro, 4000R, 4000R Pro 306 nm UV excitation with ethidium bromide (standard orange, 600 nm) emission filter
Syngene G:Box, InGenius, U:Genius Mid rage UV excitation with ethidium bromide emission filter
Biometra BioDocAnalyze Mid-range (312 nm) UV excitation with ethidium bromide emission filter
Bio-Rad All Gel Doc, ChemiDoc, VersaDoc imagers, EXQuest spot cutter Standard UV (302 nm) lamp, with the 580 nm bandpass for ethidium bromide for Molecular Imager Gel Doc XR+ and ChemiDoc XRS+ systems, or either the 520 nm longpass filter or the 605 nm bandpass filter for Molecular Imager VersaDoc MP systems
Imager Compatibility Chart
Stain UV system White Light system Molecular Imager Gel Doc system Molecular Imager ChemiDoc system Molecular Imager VersaDoc system EXQuest spot cutter Molecular Imager PharosFX system Typhoon system
Oriole +++ +++ +++ +++ +++
Flamingo + +++ + + +++ + +++ +++
Krypton + ++ + + ++ + ++ ++
Deep Purple + ++ + + ++ + ++ ++
SYPRO Ruby +++ ++ ++ ++ +++ ++ +++ +++

 +++ most compatible  |  + least compatible  |  — not compatible 

2-D Gel Stained with Flamingo and SYPRO Ruby Stain

Comparison of 2-D gel staining with Flamingo vs. SYPRO Ruby. Total E.coli protein samples were separated by 2-D electrophoresis. Separation in first dimension was performed using 11 cm pH 3–10 NL ReadyStrip IPG strips. All gel images were generated with the Molecular Imager FX system using default instrument settings.

Linearity of Staining

Linear dynamic range of staining intensity. Gels containing dilution series of protein standards were stained either with Flamingo™ or SYPRO Ruby and scanned using the PharosFX™ system. The Flamingo-stained gel was scanned using the instrument setting for medium-intensity samples (45% maximum PMT voltage). SYPRO Ruby gels were scanned using the instrument setting for low-intensity samples (55% maximum PMT voltage). The total pixel intensity following background subtraction associated with the ovalbumin band was determined across the entire visible range of the dilution series. Fluorescence is given in arbitrary relative units. Results are graphed over three concentration ranges: high range, 30–960 ng; mid range, 2–60 ng; low range, 0.125–4 ng.
Excitation and Emission Spectra
Oriole excitation and emissions

Fluorescence excitation and emission spectra of Oriole stain. Oriole stain has its excitation maximum at 270 nm and emission maximum at 604 nm, making it compatible with UV-based imagers. , Excitation spectrum; , Emission spectrum.

Flamingo excitation and emissions

Excitation (A) and Emission (B) spectra for Flamingo fluorescent gel stain in the presence of protein (denatured carbonic anhydrase).

SYPRO Ruby fluorescence excitation and emissions spectra

Fluorescence excitation and emission spectra of SYPRO Ruby protein gel stain.

Comparison of Different Stains

Gel images generated by laser staining. Gels containing a dilution series of protein standards were stained with Flamingo, SYPRO Ruby, Deep Purple or Krypton stains and scanned using Molecular Imager PharosFX system. The images were transformed to show the lowest possible visual limit of sensitivity. Only the portion of each gel showing protein loads of 1 ng or lower is shown.

2-D Gel Stained with Oriole™ Stain

2-D gel stained with Oriole™ stain. E. coli protein (40 µg) was run on an 11 cm pH 5–8 ReadyStrip™ IPG strip for the first dimension and Tris-HCl 8–16% Criterion gel for the second dimension. The gel was stained with Oriole stain and was imaged on the Molecular Imager® VersaDoc™ imaging system.

Basic Staining Protocols

Basic staining protocols for SYPRO Ruby and Oriole™ stains. All steps were carried out at room temperature.

Processing Step SYPRO Ruby Stain Oriole Stain
Fixing 1 30 min None
Fixing 2 30 min None
Staining Overnight 90 min
Wash 30 min None
Total time ~18 hr 90 min
MS-Based Protein Identification

Summary of data comparing MS-based protein identification between Oriole™ and SYPRO Ruby stain. The data were evaluated with respect to three different parameters — the number of peptides matched to known tryptic peptides, the percentage of the full sequence represented by the identified peptides, and the MASCOT score.

Protein MASCOT Score Approximate Quantity # of Peptides Matched % Coverage MASCOT Score
  in Gel Plug, ng Oriole SYPRO Ruby Oriole SYPRO Ruby Oriole SYPRO Ruby
β–Galactosidase 160 52 40 62 45 212 82
  40 42 31 48 34 107 67
  10 18 19 16 18 79 33
Ovalbumin 160 25 23 48 71 32 107
  40 10 8 20 29 28 45
  10 15 11 48 32 50 26
  160 12 11 68 68 72 48
  40 9 15 53 58 53 56
  10 13 9 68 53 53 24
Sum   196 167 431 408 686 488

Fluorescent protein gel staining, when combined with an appropriate imager, is a superior approach for protein analysis compared to Coomassie and silver staining. Fluorescent gel stains offer advantages such as broad linear range, higher sensitivity, and mass spectrometry compatibility. Bio-Rad offers three types of fluorescent protein gel stains, each optimized for your imager requirements:

Oriole™ fluorescent gel stain — An easy-to-use, fast, sensitive, and highly linear protein gel stain for visualization and quantitation of proteins in SDS-PAGE gels. The simple protocol requires a one-step staining process without prior fixing. Since destaining is not required, protein samples can be accurately visualized and quantitated in less than 2 hours. The excitation and emission characteristics of Oriole make it ideal for UV-based imagers.

Flamingo™ fluorescent gel stain — This 10x solution contains a novel dye that fluoresces when bound to denatured proteins. This easy-to-use gel stain can be used in both 1-D and 2-D electrophoretic applications and can detect down to 0.5 ng of protein. The staining procedure is a simple two-step protocol that can be completed in as little as 5 hours. The steps are not time sensitive and destaining is not required. Stained gels may be stored in the dark at 2–8°C for up to 6 months without significant loss of imaging sensitivity. Flamingo fluorescent gel stain is compatible with mass spectrometry. Gels stained with Flamingo fluorescent gel stain can also be visualized with a variety of fluorescence imaging systems, but it is particularly optimized for laser-based fluorescence scanners such as the Molecular Imager® PharosFX™ system.

SYPRO Ruby protein gel stain — The most widely used gel stain for 2-D electrophoresis, ready-to-use SYPRO Ruby protein gel stain is compatible with mass spectrometry and Edman-based sequencing applications. It can be visualized with UV or blue light transilluminators or laser scanners.

Oriole Fluorescent Gel Stain

161-0495
200 ml, 1x Oriole fluorescent gel stain solution

List Price:   $77.00
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Hazardous  
Oriole Fluorescent Gel Stain

161-0496
1 L, 1x Oriole fluorescent gel stain solution

List Price:   $169.00
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Oriole Fluorescent Gel Stain, Kit

161-0497
5 L kit, materials sufficient for 5 L of 1x Oriole fluorescent stain solution

List Price:   $779.00
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Flamingo Fluorescent Gel Stain

161-0490
20 ml, 10x Flamingo fluorescent gel stain solution

List Price:   $63.00
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Flamingo™ Fluorescent Gel Stain

161-0491
100 ml, 10x Flamingo fluorescent gel stain solution

List Price:   $179.00
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Flamingo Fluorescent Gel Stain

161-0492
500 ml, 10x Flamingo fluorescent gel stain solution

List Price:   $855.00
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SYPRO Ruby Protein Gel Stain

170-3126
200 ml, 1x solution, ready-to-use fluorescent gel stain

List Price:   $85.00
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SYPRO Ruby Protein Gel Stain

170-3125
1 L, 1x solution, ready-to-use fluorescent gel stain

List Price:   $295.00
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SYPRO Ruby Protein Gel Stain

170-3138
5 L, 1x solution, ready-to-use fluorescent gel stain

List Price:   $1,019.00
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Number Description Options
5991 Oriole Fluorescent Gel Stain: A Novel Fluorescent Stain for a Simpler 2-D PAGE to MALDI Workflow, Rev A Click to download
2533 SYPRO Ruby Protein Gel Stain Flier, Rev E Click to download
5989 Imaging Fluorescently Stained Gels with Image Lab Software Quick Start Guide, Rev A Click to download
5705 Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains, Rev A Click to download
5346 Flamingo Fluorescent Gel Stain Product Information Sheet, Rev A Click to download
10003321 Instruction Manual, Flamingo Fluorescent Gel Stain, Rev A Click to download
4006173 Instruction Manual, SYPRO Ruby Protein Stains, Rev B Click to download
10017295 Instruction Manual, Oriole Fluorescent Gel Stain, Rev B Click to download
5900 Oriole Fluorescent Gel Stain PIS, Rev A Click to download
5754 Comparison of SYPRO Ruby and Flamingo Fluorescent Gel Stains With Respect to Compatibility With Mass Spectrometry, Rev A Click to download
5921 Oriole Fluorescent Gel Stain: Characterization and Comparison with SYPRO Ruby Gel Stain, Rev A. Click to download