Oriole™ Fluorescent Gel Stain Instruction Manual Catalog # 161-0495, 1x solution, 200 ml 161-0496, 1x solution, 1 L 161-0497, kit for 5 L For technical support call your local Bio-Rad office. In the U.S. call 1-800-424-6723.
Table of Contents Section 1: Introduction and General Information 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Introduction Product Description Storage Materials and Equipment Required but Not Supplied Reagents Required but Not Supplied Safety Considerations Disposal Considerations Fluorescence Characteristics Section 2: Instructions 2.1 2.2 2.3 2.
Section 3: Technical Information 14 3.1 3.2 3.3 3.4 3.5 14 15 15 16 17 Sensitivity of Staining — 1-D Gels Compatibility with Mass Spectrometry Protein-to-Protein Variability Dynamic Range 2-D Gel Staining Section 4: Troubleshooting 18 Section 5: Product Information 21 5.1 Oriole™ Fluorescent Gel Stain 5.
Section 1 Introduction and General Information 1.1. Introduction Oriole™ fluorescent gel stain is an easy to use, rapid, and sensitive stain for visualization and quantitation of proteins separated by SDS-PAGE. The product is available in three configurations. The 200 ml and 1 L sizes are provided ready to use. The product is also available as a kit containing components to make 5 L of ready to use staining solution.
1.2. Product Description Oriole fluorescent gel stain comes in three package configurations. The 200 ml size — fully diluted and ready to use; provides enough stain for four mini format Mini-PROTEAN® gels (~8.6 x 6.8 cm), or two midi format Criterion™ gels (13.3 x 8.7 cm). The 1 L size — fully diluted and ready to use; provides enough stain for 20 Mini-PROTEAN gels (~8.6 x 6.8 cm), ten Criterion gels (13.3 x 8.
before using. Avoid prolonged exposure to temperatures greater than 37°C and protect from light. 1.4.
1.6. Safety Considerations Oriole fluorescent gel stain is a dilute solution of a fluorescent dye. The working solution is flammable and should be handled in a manner that prevents exposure to open flame or sparks. 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.
Fluorescence, normalized 120 100 80 60 40 20 0 200 300 400 500 600 Wavelength, nm 700 800 Fig. 1. 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.
Section 2 Instructions 2.1. General Considerations Best results are obtained by using clean technique. Any dust or dirt transferred to the surface of the gel may appear in the fluorescence image as smudges or speckles. Oriole™ 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.
same general order as silver stain or other fluorescent protein stains. Oriole fluorescent gel stain has a wide dynamic range, and variability in the amount of protein to be visualized can be accommodated simply by varying the exposure settings during imaging. As a general rule, the maximum quantity of protein recommended for visualization with Oriole fluorescent gel stain is 1–2 µg for individual proteins and 10–20 µg for complex mixtures on 1-D gels.
Molecular weight standards that have been prestained with a visible dye such as Precision Plus Protein™ All Blue, Dual Color or Kaleidoscope™ prestained standards do not stain with Oriole fluorescent gel stain and cannot be imaged by fluorescence in gels stained with Oriole stain. We recommend the use of unstained protein standards on gels to be stained with Oriole stain. 2.2. Stain Solution Preparation The 200 ml and 1 L configurations are provided ready to use.
NOTE: Use only methanol in preparing staining solution from the 5 L kit. The use of water, ethanol, or other solvents will result in poor staining performance. Recommended Stain Volume Gel Size Volume of Stain Solution per Gel Ready Gel® or Mini-PROTEAN gel (8.6 cm × 6.8 cm) Criterion gel (13.3 cm × 8.7 cm) 50 ml 100 ml PROTEAN II gel (16 cm × 16 cm or 16 cm x 20 cm) 250 ml PROTEAN Plus gel (25.6 cm × 23 cm) 500 ml 2.3. Gel Staining NOTE: Do not fix or wash gel prior to staining.
2. Stain for 90 min for maximum sensitivity. 3. Cover the tray to exclude light if the gel is stained longer than 90 min. NOTE: Best results are obtained if the gels are left in staining solution no longer than 2 hr. 4. Transfer gel to water prior to imaging. This step prevents exposure of the imaging equipment to moderately corrosive staining solution. NOTE: Destaining is not necessary. Staining intensity persists when the gel is stored in water.
2.4. Gel Imaging Gels stained with Oriole stain are visualized using UV light excitation. Bio-Rad Molecular Imager Gel Doc XR+, Molecular Imager ChemiDoc XRS+, EXQuest spot cutter, VersaDoc MP 4000, or VersaDoc MP 5000 systems are recommended for imaging gels stained with Oriole stain. If the imaging equipment has no preprogrammed imaging function for Oriole fluorescent gel stain, the imaging setting for SYPRO Ruby stain or ethidium bromide that uses UV transillumination is recommended.
Manufacturer Imaging system Recommended settings Bio-Rad Gel Doc, ChemiDoc, For Gel Doc and ChemiDoc Laboratories VersaDoc, EXQuest systems, use Standard spot cutter systems (302 nm) UV lamp with Standard Filter (580 nm bandpass for ethidium bromide).
Manufacturer Imaging system Recommended settings UVP Midrange (302 nm) UV excitation with ethidium bromide red emission filter BioDoc-It, VisiDoc-It, DigiDoc-It, MultiDoc-It, Photo Doc-It, BioSpectrum, EC3 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,
Section 3 Technical Information 3.1. Sensitivity of Staining — 1-D Gels The dye in Oriole™ stain is highly fluorescent and binds tightly to proteins. Background staining is low, and the limit of sensitivity is generally below 1 ng. Fig. 2. Unadjusted image of a gel stained with Oriole stain.
4 2 1 0.5 0.25 0.125 ng/band Fig. 3. Image of the gel stained with Oriole stain, adjusted to show the limit of sensitivity. The image from the previous figure was inverted, cropped to show protein loads ≤ 4 ng, and adjusted to show the limit of sensitivity. 3.2. Compatibility With Mass Spectrometry Oriole fluorescent protein gel stain is fully compatible with downstream proteolysis and mass spectrometric analysis. 3.3.
3.4. Dynamic Range Oriole fluorescent gel stain has a dynamic range of up to three orders of magnitude. This allows protein quantitation in samples of varying concentration over a wide range of relative abundance. Log (fluorescence), arbitrary units 4.5 4 3.5 3 2.5 2 0 0.5 1 1.5 2 Log carbonic anhydrase, ng 2.5 Fig. 4. Linearity of Oriole stain.
3.5. 2-D Gel Staining Oriole fluorescent gel stain is ideal for staining 2-D polyacrylamide gels. Clear background-free images are obtained without interference from CHAPS, carrier ampholytes, or other components of the first-dimension separation. Fig. 5. 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 Criterion Tris-HCl 8–16% gel for the second dimension.
Section 4 Troubleshooting Problem Possible Cause Remedy Bands or spots No protein on gel not visible Verify that there is actually protein on the gel by staining with another method such as Bio-Safe™ Coomassie stain. Malfunctioning imaging system Check instrument manual for troubleshooting, or contact the imaging instrument manufacturer. Poor staining sensitivity Stain sensitivity maximizes after 90 min.
Problem Possible Cause Poor staining Reuse of the stain sensitivity Remedy Reuse of Oriole stain is not recommended. Use of Use only methanol and nonrecommended provided diluent. solvent for the 5 L kit Fixing or washing the gel prior to staining Do not fix or wash gel prior to staining.
Problem Possible Cause Remedy High or uneven background staining Reagent impurities when preparing stain solution from the 5 L kit Use high quality reagents. Speckles or blotches in the gel image Particulate material from reagents, staining tray, dust, or gloves Make sure that the staining trays are thoroughly cleaned. 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.
Section 5 Product Information 5.1. Oriole Fluorescent Gel Stain Catalog # Description 161-0495 161-0496 161-0497 Oriole™ Fluorescent Gel Stain, 1x solution, 200 ml Oriole Fluorescent Gel Stain, 1x solution, 1 L Oriole Fluorescent Gel Stain, kit for 5 L 5.2.
Bio-Rad Laboratories, Inc. is licensed by Invitrogen Corporation to sell SYPRO products for research use only, under U.S. patent 5,616,502. AlphaImager and FluorChem are trademarks of Alpha Innotech Corporation. BioDoc-It, BioSpectrum, DigiDoc-It, Doc-It, and MultiDoc-It are trademarks of UVP, LLC. Coomassie is a trademark of BASF Aktiengesellschaft. Ettan, ImageQuant, Typhoon, and Storm are trademarks of GE Healthcare Group Companies.Odyssey is a trademark of LI-COR, Inc.
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