AdvanStain Scarlet user manual
Contents
1. page 7 page 8 Advan8tain Scarlet 9 Detailed Protocol Blot Plaining Step 1 Washing e Following transfer wash blot for 5 min in water e Proceed to PVDF 2 or Nitrocellulose 3 protocol 2 PVDF protocol 2a Staining e Place blot protein side down in Stain Solution e Stain blot with gentle rocking for 15 30 min 2b Acidification e Place blot in Fix Solution and incubate with gentle rocking for 5 min 2c Wash e Rinse blot 3 times with 100 ethanol for 2 3 min each until green background on blot has been completely removed 2d Drying e Hang blot from a peg or dry on wire mesh to allow blot to dry evenly e Allow blot to dry completely before imaging 3 Nitrocellulose Protocol 3a Staining e Place blot protein side down in Stain Solution e Stain blot with gentle rocking for 15 30 min D 05023 028 Notes For best results run the buffer front off the base of the gel during electrophoresis prior to transfer Do not allow membrane to dry during staining e For all steps use 50 ml for small blots 400 ml for large e Prepare Stain Solution Allow AdvanStain Scarlet Dye to warm to room temperature Mix thoroughly For small blots dilute 125 ul AdvanStain Scarlet Dye in 50 ml Stain Buffer Mix well For large blots dilute 1 ml of AdvanStain Scarlet Dye in 400 ml Stain Buffer Mix well Blot will appear green Methanol may used instead of ethanol2. e Prepare Stain Solution Allow AdvanStain Scarlet Dye to warm to room temperature Mix thoroughly For small blots dilute 125 ul AdvanStain Scarlet Dye in 50 ml Stain Buffer Mix well For large blots dilute 1 ml of AdvanStain Scarlet Dye in 400 ml Stain Buffer Mix well 9 Detailed Protocol Plaining Blots continued Step Notes 3b Acidification e Place blot in Fix Solution and incubate with gentle rocking for 5 min Blot will appear green 3c Washing e Wash blot 1 time in Wash Solution for 5 min with gentle rocking e Wash blot 2 times in high purity water for 5 min with gentle rocking 3d Drying 1 e Allow blot to dry completely before imaging 0 Vestaining AdvanStain Scarlet staining is reversible and the stain may be removed for subsequent analysis such as Western blotting 1 To destain while minimizing protein loss Wash blot overnight in 50 mM ammonium carbonate 2 To rapidly destain PVDF membranes Wash blot with 50 acetonitrile containing 30 mM ammonium carbonate for 15 min 3 To rapidly destain nitrocellulose membranes Wash blot with 50 ethanol or methanol containing 50 mM ammonium carbonate for 15 min 4 To rapidly destain protein gels Wash blot with 50 ethanol or methanol containing 50 mM ammonium carbonate for 15 min to 1 hour www advansta com page 9 page 10 AdvanStain Pcarlet ae P orage Gels may be stored at 4 C in 1 citr
3. Advan tain Scarlet Fluorescent total protein stain for gels and blots For Catalog Numbers K 11072 B50 AdvanStain Scarlet Kit 5 ml dilutes to 1 L K 11072 C25 AdvanStain Scarlet Kit 25 ml dilutes to 5L Advansta Corporation 1505 Adams Drive Suite B1 Menlo Park CA 94025 Tel 650 325 1980 Fax 650 325 1904 Email sales advansta com Product information www advansta com AdvanStain_Scarlet February 24 2014 D 05023 028 www advansta com Advan8tain Scarlet Important Information The following instructions are for use with AdvanStain Scarlet total protein stain catalog numbers K 11072 250 K 11072 B50 and K 11072 C25 Please see the Kit Contents section for details Ptorage Information Store AdvanStain Scarlet Dye in a freezer at 15 C to 30 C in the original brown bottle provided and protect from light The AdvanStain Scarlet Powder A and Powder B are stable at room temperature for one year Warnings and Precautions e AdvanStain Scarlet total protein stain is for research use only e Always wear gloves when handling membranes and reagents e Refer to MSDS for additional safety information e The product is guaranteed to be free of manufacturer defect and to function as described when the enclosed protocol is followed by properly trained personnel Please see the Warranty section for more information D 05023 028 Table of Contenti Section 1 Kit Contents 2 Shipping and Storage Con
4. below 27 C is acceptable if the total dispatch time is no longer than 5 days Upon receipt store AdvanStain Scarlet Dye in a freezer at 15 C to 30 C in the original brown bottle provided and protect from light AdvanStain Scarlet Powder A and Powder B may be stored at room temperature in a dry location 3 Additional Materials Required e High purity water distilled Milli Q or equivalent e 100 ethanol e Staining tray e Shaking or rocking platform D 05023 028 4 About AdvanPtain Pcarlet AdvanStain Scarlet is based on epicocconone a small naturally occurring fluorescent compound that reversibly binds to lysine arginine and histidine residues in proteins and peptides to yield an intensely red fluorescent product This unique mechanism provides sensitive quantification of proteins in 1D and 2D gels of all chemistries on both PVDF and nitrocellulose blots and provides unparalleled compatibility with Mass Spectrometry 5 Excitation and Enussion Yoectra Optimal excitation wavelengths for AdvanStain Scarlet are 405 or 500 nm Compatible excitation light sources include green 543 532 nm blue 488 nm violet 405 nm or UV 302 365 nm The maximum emission wavelength of AdvanStain Scarlet stain is 610 nm regardless of what excitation source is used 610 nm band pass or 560 long pass filters may be used The excitation and emission spectra of AdvanStain Scarlet can be seen in Figure 1 1 2 Normal
5. purity water and wash in Wash Solution for 30 min with gentle rocking 4 Acidification e This step can be repeated or extended to overnight to e Remove gel from Wash reduce background staining Solution and place in Fix If performing this step overnight protect the gel from Solution light e Incubate in Fix Solution for 30 min with gentle rocking 5 Imaging Compatible excitation sources include green 543 Detect fluorescence at 532 nm blue 488 nm violet 405 nm or UV 610 nm using standard 302 365 nm fluorescence scanners and Detect fluorescence using a 610 nm band pass or CCD camera systems For 560 nm long pass filter recommended imaging settings refer to Table 3 D 05023 028 8 cm x 11 cm x 1mm 100 ml 50 ml 250 uL 100 ml 100 ml mini gels 13 3 cm x 8 7 cm x 1 mm 200 ml 100 ml 500 uL 200 ml 200 ml small format 2D gels Table 2 Volumes of Solutions For Different Gel Sizes Laser scanner Green Orange long 532 nm light pass 560 nm filter or red 610 nm filter CD imager with Long Orange long transilluminator wavelength pass filter UV 302 365 nm or black light blue lamp Ettan DIGE Imager Green Orange For multiplex applications GE Healthcare 540 25 nm 595 25 nm violet excitation filter 390 20 light source filter nm and orange emission filter will avoid cross talk with Cy2 and Cy3 Table 3 Recommended Imaging Conditions for Different Imaging Systems www advansta com
6. sheets 1 ml 5 ml 1 ml 5 ml 15 Warranty This product is warranted to be free of defects of material or workmanship and to perform as described in the published specifications when stored according to the documentation included with the product and used according to the accompanying instruction manual by appropriately trained personnel If the product is found to have a defect upon first use and within 30 days of shipment the product may be replaced This warranty extends only to the original purchaser of the product There is no obligation to replace the product as a result of misuse improper storage or negligence of the buyer 16 User Notes Copyright 2012 Advansta All rights reserved AdvanStain Scarlet Afyon WesternBright and the Advansta logo are trademarks of the Company All other trademarks service marks and trade names appearing in this brochure are the property of their respective owners www advansta com page 13 page 14
7. ditions 3 Additional Materials Required 4 About AdvanStain Scarlet 5 Excitation and Emission Spectra 6 Overview of AdvanStain Scarlet Gel Staining Protocol 7 Preparation of Solutions 8 Detailed Protocol Gel Staining 9 Detailed Protocol Blot Staining 10 Destaining 11 Storage 12 Troubleshooting and FAQ 13 References 14 Related Products 15 Warranty 16 User Notes www advansta com Page O ON Oo A A WW Co Se CG gt A A OO N gt gt page 1 page 2 Advan8tain Pcarlet 1 Kit Contents Catalog Number K 11072 B50 AdvanStain Scarlet total protein stain 5 ml Item Description Quantity R 04016 C10 AdvansStain Scarlet Powder A 4x10 1g R 04017 C23 AdvanStain Scarlet Powder B 23 4 g R 03016 B50 AdvanStain Scarlet Dye 5 ml AdvanStain Scarlet Stain 5 ml kit is sufficient for staining twenty SDS PAGE mini gels 8 cm x 11 cm or four full size 2 D gels 17 cm x 17 cm Catalog Number K 11072 C25 AdvanStain Scarlet total protein stain 25 ml Item Description Quantity R 04016 C10 AdvanStain Scarlet Powder A 20x 10 1g R 04017 C23 AdvanStain Scarlet Powder B 5x 23 4g R 03016 C25 AdvanStain Scarlet Dye 25 ml AdvanStain Scarlet Stain 25 ml kit is sufficient for staining one hundred SDS PAGE mini gels 8 cm x 11 cm or twenty full size 2 D gels 17 cm x 17 cm 2 Phipping and P orage Conditions Product may be shipped refrigerated or frozen on blue ice or dry ice Shipping at ambient temperature
8. e Extend washing time e Filter buffers to remove dust or precipitates e Protect gel from airborne particles 13 References 1 Bell P J L and Karuso P 2003 Epicocconone a novel fluorescent compound from the fungus Epicoccum nigrum Journal of the American Chemical Society 125 9304 9305 Coghlan D R Mackintosh J amp Karuso P 2005 Mechanism of reversible fluorescent staining of protein with Epicocconone Organic Letters 7 2401 240 Mackintosh J A Veal D A and Karuso P 2005 FluoroProfile a fluorescence based assay for rapid and sensitive quantification of proteins in solution Proteomics 5 4673 4677 Malmport E Mackintosh J Ji H Veal D amp Karuso P 2005 Visualization of proteins electro transferred on Hybond ECL and Hybond P using Deep Purple Total Protein Stain GE Healthcare Life Science News 19 12 13 Mackintosh J A Choi H Y Bae S H Veal D A Bell P J Ferrari B C van Dyk D Verrills N M Paik Y K amp Karuso P 2003 A fluorescent natural product for ultra sensitive detection of proteins in 1 D and 2 D gel electrophoresis Proteomics 3 2273 2288 Tannu N S Sanchez Brambila G S Kirby P Andacht T M 2006 Effect of staining reagent on peptide mass fingerprinting from in gel trypsin digestions A comparison of Sypro Ruby and Deep Purple Electrophoresis 27 3136 3143 Nock C M Ball M S White I R S
9. ic acid and protected from light For extended storage up to 6 months add AdvanStain Scarlet Dye to the storage solution at 1 200 Prior to imaging rinse gels 2 x 15 min in Wash Solution Incubating in Fix Solution for 15 minutes can reduce background Blots may be stored dry in the dark at room temperature 12 Troubleshosting amp FAQ Problem High background No or low signal Negative staining Speckled background D 05023 028 Possible Solutions e Handle gels with clean non powdered gloves and avoid contamination with dust e Ensure concentrated AdvanStain Scarlet Dye was brought to room temperature and thoroughly mixed prior to dilution e Ensure stain was thoroughly mixed into Stain Buffer before adding to gel e Stain only one gel per tray e Use high purity water distilled Milli Q or equivalent e Check pH during staining step pH should be between 9 5 and 10 5 Carry over acid from the fixation step can result in poor staining e Stain may fade with long exposure times and associated heating on CCD based instruments e Ensure stain concentrate was brought to room temperature and mixed thoroughly before dilution e Staining for over 2 hours in alkaline conditions destabilizes proteins and leads to diffusion of protein bands from the gel matrix e Use high quality SDS in the preparation and running of the gel e Extend fixation time to overnight e Use correct volumes of Fix and Wash Solutions
10. ized Excitation i Normalized Emission Intensity 300 350 400 450 500 550 600 650 700 750 800 Wavelength nm Figure 1 Excitation and Emission Spectra of AdvanStain Scarlet Dye www advansta com page 3 page 4 Advan8tain Scarlet 6 Overview of AdvanPtain Pcarlet Gel Plaining Protocol Fixation Incubate gel in Fix Solution 1 hour to overnight ii Staining Incubate gel in Stain Solution 1 to 2 hours ii Washing Rinse gel with high purity water and incubate in Wash Solution 30 to 45 minutes l Acidification Incubate gel in Fix Solution 30 minutes to overnight l Image D 05023 028 7 Preparation of Polutions Before staining prepare Fix Stain and Wash solutions as described below These solutions are stable for up to 1 year when stored at room temperature Precipitates or dust present in the solutions will result in speckling on gels If observed filter solutions before use The amount of reagents in each packet of AdvanStain Powder A or B is sufficient to prepare 1 L of solution Do not split the packets Once a packet is opened the entire contents should be used For preparation of larger volumes use more than one pouch Fix Solution Add contents of one AdvanStain Scarlet Powder A packet 10 1 g to 850 ml of high purity water in a 1 L bottle Mix until dissolved Add 150 ml 100 ethanol and mix thoroughly Stain Buffer Add contents of one AdvanStain Scarlet Powder B packe
11. kehel J M Bill L and Karuso P 2008 Mass Spectrometric Compatibility of Deep Purple and SYPRO Ruby total protein stains for high throughput proteomics using large format two dimensional gel electrophoresis Rapid Communications in Mass Spectrometry 22 881 886 Ball M S Karuso P 2007 Mass Spectral Compatibility of Four Proteomics Stains Journal of Proteome Research 6 4313 4320 www advansta com page 11 page 12 Advan8tain Pcarlet 14 Related Products Catalog Number Product K 02101 010 K 02101 025 K 12045 C20 K 12045 D20 K 12045 D50 L 08001 010 L 08002 010 L 08003 010 R 03018 B10 R 03018 B50 R 03019 B10 R 03019 B50 D 05023 028 Afyon SDS PAGE Sample Preparation Kit Afyon SDS PAGE Sample Preparation Kit WesternBright ECL Western Blotting HRP Substrate trial kit size WesternBright ECL Western Blotting HRP Substrate for 2000 cm membrane WesternBright ECL Western Blotting HRP Substrate for 5000 cm membrane Low Fluorescence Western Membrane PVDF 7x9 cm Nitrocellulose Transfer Membrane 0 45 um 7x9 cm Nitrocellulose Transfer Membrane 0 22 um 7x9 cm Non reducing protein sample loading buffer 2X Non reducing protein sample loading buffer 2X Reducing protein sample loading buffer 2X Reducing protein sample loading buffer 2X Size 10 rxns 25 rxns 20 ml 200 ml 500 ml 10 sheets 10 sheets 10
12. t 23 4 g to1L of high purity water in a 1 L bottle Mix until completely dissolved Wash Solution Mix 850 ml high purity water and 150 ml 100 ethanol in a 1 L bottle www advansta com page 5 page 6 Advan8tain Scarlet 8 Detailed Protocol Gel Ptainino 1 Fixation e For gels thicker than 1 mm or backed gels increase Fix gel in Fix Solution for a the fixation time to 1 5 hr minimum of 1 hr with gentle The fixation time can be extended to overnight rocking For correct volumes at each step refer to Table 2 2 Staining e To prepare Stain Solution Allow AdvanStain Scarlet e Prepare the Stain Solution Dye to warm to room temperature Mix thoroughly immediately prior to then dilute 1 part AdvanStain Scarlet Dye in 200 parts staining Stain Buffer Mix well Refer to Table 2 for volumes of Remove gel from Fix solutions used for different gel sizes Solution and place in Stain Stain Solution will degrade over time Prepare only as Solution minimizing carry much as is needed and use immediately over of the fixing solution Increase staining time to 1 5 hours for gels 1 5 mm Stain gel for 1 hour with thick or backed gels Extending the staining time to 2 gentle rocking hours will not affect results DO NOT stain for longer than 2 hours 3 Washing e For 1 5 mm thick gels or gels with high background e Remove gel from Stain fluorescence increase washing time to 45 min Solution rinse with high
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