WesternBrightTM MCF
Contents
1. 12 Warranty ght MCF This product is warranted to be free of defects of material or workmanship and to perform We S te p nBr igh t as described in the published specifications when stored according to the documentation included with the product and used according to the accompanying instruction manual Quantitative multi color fluorescent Western by appropriately trained personnel If the product is found to have a defect upon first use r i and within 30 days of shipment the product may be replaced This warranty extends only blotting kit 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 Copyright 2010 Advansta All rights reserved WesternBright AdvanWash AdvanBlock and the Advansta logo are trademarks of the Company All other trademarks service marks and tradenames appearing in this brochure are the property of their respective owners For Catalog Yundber K 12021 010 WesternBright MCF fluorescent Western blotting kit Advansta Corporation 1455 Adams Drive Ste 1160 Menlo Park CA 94025 Tel 650 325 1980 Fax 650 325 1904 Email sales advansta com Product information www advansta com WesternBright_MCF December 6 2012 D 05014 012 www advansta com WesternBright MCF Important Informatiu Table of Contents The following instructions are for use with the WesternBright MCF fluorescen2. Western blot The kit contains two secondary antibody conjugates each pre labeled with a different fluorescent reporter Fluorescent reporters utilized in the WesternBright MCF kit are phycobiliproteins highly fluorescent proteins isolated from cyanobacteria and eukaryotic algae that live in environments with very low natural illumination These proteins are components of the natural photosynthetic system of these organisms The function of these proteins is to harvest light at all available spectral bands and transfer its energy to chlorophyll with a fluorescent energy transfer mechanism Millions of years of evolution created several different fluorophores with various absorption spectra to accept all available light and transfer it to an appropriate acceptor with very high quantum efficiency R phycoerythrin RPE and allophycocyanin APC are two variants commonly used as reporters in FACS analysis due to their good spectral separation and compatibility with laser light sources utilized in cell sorters The excitation and emission spectra of RPE are shown in Figure 1 and for APC in Figure 2 Until now fluorescent reporters in general and phycobiliproteins in particular were not widely used for Western blotting applications for various reasons Membranes typically used for Western blotting had high levels of autofluorescence availability of appropriate imaging instruments was very limited and the acquisition time required to image a typical blot w
3. 0 21 1123 1144 one more time The membrane can be dried after this point However lower protein signal levels are expected with 4 Bolt M W Mahoney P A High efficiency blotting of proteins of diverse sizes following prolonged storage sodium dodecyl sulfate polyacrylamide gel electophoresis Anal Biochem 1997 May 1 247 2 185 192 We have a blocking We advise against using alternate blocking or washing solutions solution and or wash Alternate components may result in a higher background or solution we typically reduced sensitivity When the membrane is dried after using 11 Related Products used can we integrate other blocking solutions the fluorescent intensity and stability those components into _ of fluorophores may be reduced to a greater extent However Catalog Number Product Size the kit methods if your primary antibodies have very significant non specific R 05051 250 APC goat anti rabbit IgG conjugate 250 ul cross reactivity with other proteins or with IgG and you must use your blocker you can use the AdvanBlock PF blocking R 05052 250 RPE goat anti mouse IgG conjugate 250 ul solution provided with the kit as a base to prepare your specific R 03023 D20 AdvanBlock PF protein free 5x blocking antibody diluent solution If you intend to use your blocker we suggest using AdvanBlock PF for the membrane blocking step solution 200 ml 2 4 of the protocol and use your specific blocker to prepare R 03024 D50 AdvanWash 1
4. 0x washing solution 500 ml the primary antibody incubation solution step 2 5 R 01038 020 Avant Buffer Pouches PBS 20 pouches R 01039 020 Avant Buffer Pouches TBS 20 pouches We do not detect signal Check to see if the transfer was successful by using a protein on the blot standard if this is positive check the imaging system to confirm R 01037 020 E w i l Avant Buffer Pouches Tris Glycine the correct excitation and emission settings Typically imaging channels optimized for Cy3 and Cy5 dyes work well with the PAGE Cunning Huer ey oucnye WesternBright conjugates If you are trying to detect small R 01036 020 Avant Buffer Pouches Tris Glycine amounts of a target protein try to increase the concentration of SDS PAGE running buffer 20 pouches your primary antibody first If this is unsuccessful also increase the concentration of the secondary conjugates L 07031 005 Incubation Tray red 7x5 cm 5 pack L 07032 005 Incubation Tray black 9x6 cm 5 pack Our lab generally uses We suggest using the Blocking and Washing solutions supplied L 07033 005 Incubation Tray blue 11x9 cm 5 pack Tris Phosphate based with the kit to guarantee best performance However if you l buffers will this work require different buffer conditions test a small blot before L 07001 010 Background quenching sheets 10 sheets with the kit using larger quantities of kit components However if you do L 08001 010 Low fluorescence PVDF transfer membrane not use the AdvanBloc
5. Continued Step Notes 3 5 Remove the AdvanWash completely or transfer the membrane into a new clean tray 3 6 Add 10 ml of the AdvanWash to the tray with the membrane and wash for 15 minutes with gentle agitation 3 7 Wash the membrane 3 more times for 5 minutes each with gentle agitation replacing with 10 ml of fresh AdvanWash each time 4 Staining the membrane with the WesternBright secondary antibody antibody conjugates gently mix fluorescent conjugates by inverting the tube several 4 1 During the last washing step prepare the times Do NOT vortex mixture of secondary antibody fluorescent conjugates Transfer 10 ml of the AdvanWash prepared in step 3 1 into a new 15 ml tube Add 4 ul of each of the fluorescent conjugates provided in the kit dilution factor 1 2500 4 2 After the last washing step is completed remove the AdvanWash and add the secondary conjugate mix to the tray with the membrane 4 3 Incubate the membrane with the secondary antibody conjugate mixture for 1 hour at room temperature with gentle agitation e When diluting the secondary 5 Final washing e The purpose of step 5 2 is to 5 1 Remove the incubation solution and wash the remove from the membrane membrane 3 times for 5 minutes each with Tween 20 that is present in 10 ml of the AdvanWash with gentle agitation the AdvanWash Removal of 5 2 Remove the AdvanWash and add 20 50 ml of Tween 20 stabilizes the structure PBS or TBS that does N
6. OT contain Tween 20 of the WesternBright conjugates or any other detergents and wash the and preserves the fluorescence membrane for 5 minutes with gentle agitation when the membrane dries 5 3 Remove the membrane from the solution e The membrane will not fall off with clean forceps and transfer onto a black the plastic sheet as it adheres background quenching sheet and allow the to it while still wet Do not use excess liquid to run off the membrane by any wipes or tissue to remove holding the plastic sheet with the membrane the excess liquid as this may vertically increase the background and introduce random fluorescent artifacts Minor liquid excess does not affect the results D 0501 4 012 page 11 8 Imaging The membrane can be imaged immediately after the final wash step However the highest sensitivity is achieved when the membrane semi dries of the excess liquid This typically takes between 15 and 30 minutes and the membrane changes its appearance from translucent to uniform white After this moment the fluorescence remains unchanged or increases slightly within 30 minutes to 1 hour depending on the ambient temperature and relative humidity After two hours the fluorescence may decrease 20 to 40 depending on its initial intensity and on how well Tween 20 was removed from the blot during the last washing step Review the absorption and emission spectra of the WesternBright conjugates Fig 1 and 2 to choose appropriate s
7. T with gentle agitation 6 Wash blot with 1x washing buffer e 3 x 5 min with at least 0 3 ml cm membrane each time e 1x 5 min with 20 50 ml PBS or TBS without detergent 7 Place blot on background quenching sheet and drain excess liquid 8 Image using CCD camera www advansta com page 7 page 8 WesternBright MCF 7 Detailed Protocol Step 1 Protein separation and transfer 1 1 1 2 1 3 1 4 To 1 6 1 7 Separate protein samples by polyacrylamide gel electrophoresis using a mini sized gel i e 8 x 10 cm Cut a small notch from one corner of the membrane to help properly identify the orientation and the surface side of the membrane that will contain the transferred proteins Pre wet the membrane in methanol for a few moments until the membrane is completely wet Transfer the wet membrane from methanol to purified water Milli Q quality or distilled and incubate on a rocker or shaker for at least 5 minutes Make sure that the membrane is fully immersed in water and does not float on the water s surface Transfer the membrane from water to transfer buffer and incubate for 5 minutes Assemble the transfer sandwich as required for your transfer apparatus Perform the transfer overnight at 15 V or for 1 to 2 hours at 70 V with an ice pack 2 Membrane blocking and binding of the primary antibodies 2N 22 2 3 2 4 Once the transfer is completed step 1 7 r
8. ane in water prior to blocking improves retention of transferred proteins It is very important that the side of the membrane containing transferred proteins is facing up during all incubation and washing steps Step 2 5 While the membrane is in the AdvanBlock PF prepare the incubation mixture containing your primary antibody or an appropriate mixture of primary antibodies diluted in 10 ml 1 x AdvanBlock PF Utilize the remaining 10 ml of the AdvanBlock PF prepared in step 2 2 to make the incubation mixture of the primary antibodies Gently mix the incubation solution by inverting the tube several times Do NOT vortex 2 6 After the blocking is completed step 2 4 remove the solution from the tray and add the solution of primary antibodies to the membrane 2 7 Incubate the membrane with the solution of primary antibodies for 1 hour at room temperature with gentle agitation 3 Washing the membrane 3 1 About 10 minutes before the end of the incubation step prepare 100 ml of 1 x AdvanWash Mix 10 ml of the AdvanWash concentrate provided in the kit with 90 ml of purified water Milli Q quality or distilled This amount is sufficient to complete the experiment 3 2 Add 10 ml of the AdvanWash to a new tray 3 3 After the incubation step 2 7 is completed transfer the membrane from the incubation tray into the tray with the AdvanWash prepared in step 3 2 Use clean forceps to transfer the membrane Make sure that the or
9. as relatively long But the most important reason that prevented the use of phycobiliproteins in Western blotting applications was that these proteins need to remain hydrated to maintain their high levels of fluorescence When the water evaporates from the environment phycobiliproteins completely lose their ability to fluoresce This did not create any problems for flow cytometry applications because the detection of fluorescence occurs in solution Western blot membranes however can dry quickly and lose all the water necessary to sustain fluorescence and unfortunately re hydrating the membrane does not restore the fluorescence of phycobiliproteins D 0501 4 012 Today all the above problems can be easily addressed PVDF membranes with low autofluorescence have become available there are several choices of fast and high resolution fluorescent imaging instruments laser based scanners and LED based gel documentation imagers The most difficult problem is to preserve the hydration of phycobiliproteins long enough to be able to image the blot within a reasonable period of time oe Wadana ee Absorption Fluorescence Emission We have developed the AdvanBlock PF blocking Figure 1 Absorption and emission solution to address this problem The blocking specma ol RFE GOGI Onil mouse IgG solution provided with the kit contains a component anmpecy conjugare R905052 that provides an efficient blocking of the PVDF membrane from no
10. d tank transfer method mader meod an a peli ae varies ean applications Semi dry transfer 1 Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide acceptable for use with systems do not work well for fluorescent applications as they gels to nitrocellulose sheets procedure and some applications Proc Natl Acad Sci U S the kit typically generate relatively high and non uniform background A 1979 76 9 4350 4354 especially in the channel excited with green light We found that an alternative transfer technique described by Bolt and 2 Burnette WN Western blotting electrophoretic transfer of proteins from sodium Mahoney 4 is as quick as semi dry method very economical dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic and produces superior results for fluorescent applications detection with antibody and radioiodinated protein A Anal Biochem 1981 112 2 195 203 Can we dry the blot and The best performance for the blot is attained within 2 hours l PET image n days after we _ of drying it In order to preserve the fluorescence on the dried 3 Patton Wayne F A thousand points of light The application of fluorescence detection do the Western assay blot it is important that Tween 20 is completely removed See technologies to two dimensional gel electrophoresis and proteomics Electrophoresis step 5 2 for details If necessary step 5 2 may be repeated 200
11. emove the membrane from the sandwich and place it into a tray with Milli Q or distilled water for 5 minutes Prepare 20 ml of 1x AdvanBlock PF by mixing 4 ml of the provided concentrate with 16 ml of purified water Milli Q quality or distilled Transfer 10 ml of 1x AdvanBlock PF to an incubation tray Make sure that the tray is of an appropriate size for your membrane Transfer the membrane from water into AdvanBlock PF and block the membrane for 10 minutes with gentle agitation D 0501 4 012 Notes Asemi dry transfer does not work well for fluorescent applications as it typically generates relatively high and non uniform background especially in the channel excited with green light We currently suggest using a tank transfer method for fluorescent applications Increasing the transfer time especially if performed at 70 V causes an increase in background Increasing voltage beyond 70 V to shorten the transfer time also causes an increased and non uniform background especially in the channel excited with red light Refer to Bolt et al Reference 4 for an alternative transfer technique that works extremely well for fluorescent Western blotting All incubations and washes are performed on a rocker or shaker with gentle agitation at room temperature A rocker is preferred over a shaker In our experience a rocking action mixing generates more uniform background than an orbital shaker mixing Incubation of the membr
12. etting of your imaging instrument In most cases the settings appropriate for imaging Cy3 and Cy5 fluorescent dyes will work well for imaging WesternBright conjugates 9 Troubleshooting amp FAQ Western blotting can require substantial optimization due to the multiple steps involved The correct amount of protein to load on the gel and the best dilutions of primary and secondary antibodies must be determined empirically Some common questions are addressed below Question Answer What are your Follow the manufacturer s recommendations for the antibody recommendations dilution Typical antibody dilutions for primary antibodies range for primary antibody from 1 250 to 1 5000 High concentrations of primary and dilution secondary antibodies can result in high background levels on the blot Can we use Nitrocellulose membranes have very high autofluorescence nitrocellulose that makes it impossible or very difficult to detect low amounts membranes of proteins Furthermore only special low fluorescence PVDF membranes are suitable for high sensitivity fluorescent Western blotting applications In our experience the best performing type of membrane is the one provided with the kit If you require a different size membrane you can use Immobilon FL from Millipore Catalog No IPFLO0010 continued www advansta com page 12 WesternBright MCF Question Answer 10 References What kinds of We currently suggest using a standar
13. gate a APC a rabbit IgG Figure 3 Schematic imag aibo diagram of the two a bbit color assay utilized in the WesternBright MCF kit PVDF membrane proteins transferred blocking compound to the membrane www advansta com page 5 page 6 WesternBright MCF 5 Overview of the Protocol for Fluorescent Western Blots Separate proteins by PAGE and transfer to the low fluorescence PVDF membrane Block the membrane 10 minutes with AdvanBlock PF 4 Incubate with primary antibodies dilute antibodies in 1x AdvanBlock PF incubate for 1 hour 4 Wash the membrane with 1x AdvanWash e x 1 2 min rinse e x 15min 3x5min 4 Incubate with secondary antibodies dilute antibodies in 1x AdvanWash incubate for 1 hour Wash the membrane in 1x AdvanWash 8 lt 5 MIA 4 Wash the membrane in PBS or TBS without Tween 20 5 min 4 Place the membrane on a background quenching sheet and image D 05014 012 6 Quick Protocol For additional information see the detailed protocol which follows Step User Notes 1 Prepare your protein blot 2 Block membrane for 10 minutes at room temperature RT 3 Incubate blot with primary antibody for one hour at RT with gentle agitation 4 Wash blot with 1x washing buffer e 1 x quickly e 1x 15 min with 0 7 ml cm membrane e 3 x 5min with at least 0 3 ml cm membrane each time 5 Incubate blot with secondary antibody for one hour at R
14. ientation notch is in the same position so that the side of the membrane containing the transferred proteins is facing up 3 4 Rinse the membrane for 1 2 minutes with gentle agitation continued Notes The optimum dilution factor for primary antibodies must be determined experimentally by performing a titration experiment A good starting point is the dilution factor suggested by the antibody supplier In Our experience the optimal concentrations of primary antibodies are often lower and in many cases significantly lower than those suggested for common colorimetric Western blotting procedures Higher concentrations of primary antibodies often do not result in any higher specific fluorescent signals Instead higher concentrations of primary antibodies cause high background and appearance of many non specific bands Rinsing step 3 4 is more efficient if done manually instead of using a rocker or shaker Make sure that the membrane is rinsed uniformly to remove the majority of the excess primary antibody incubation solution However all agitations must be done gently to prevent any scratching or damage to the surface of the membrane Scratches or other damages to the blocked surface of the membrane create active sites that can adsorb fluorescent secondary antibody conjugates and cause fluorescent artifacts and or high background www advansta com page 9 page 10 WesternBright MCF 7 Detailed Protocol
15. k PF reagent you may lose the benefits of its protective effect on the fluorescence stability when your 9x7 cm 10 sheets membrane dries D 05014 012 www advansta com page 13 page 14
16. n specific protein binding At the same time it preserves the hydrated environment in the membrane sufficiently enough to protect the hydration of the proteins and stabilize their fluorescence This stabilization effect preserves most of the fluorescent intensity even after the membrane appears completely dry The remaining fluorescence is only 20 to 40 lower than its peak value and is sufficient to reliably detect sub nanogram amounts of Ee o i pas a ductal ise pagina Pa ange Figure 2 Absorption and emission antibodies and optimized assay conditions single spectra of APC goat anti mouse picogram detection can routinely be achieved IgG antibody conjugate R 05051 The secondary antibody conjugates provided in the kit are RPE anti mouse lgG and APC anti rabbit lgG Therefore the WesternBright kit is appropriate for experiments incorporating most readily available primary antibodies raised in mouse and or rabbit The conjugation ratio is optimized to be close to equimolar to maintain efficient diffusion rates of the conjugates that do not interfere with antibodies binding to antigens In addition the APC is also cross linked to further preserve its fluorescence under typical immuno detection conditions Absorption Fluorescence Emission A schematic representation of the two color Western blot is shown in Fig 3 secondary antibody conjugate RPE a mouse IgG AAN primary antibody mouse secondary antibody conju
17. pping and Plarage Conditions Never freeze any of the kit components Before opening the kit may be stored at 4 C WesternBright fluorescent secondary antibody conjugates must be stored at 4 C Gently mix and briefly spin the tubes before taking aliquots AdvanBlock PF solution must be stored at 4 C AdvanWash solution can be stored between 4 C and 25 C Do not dilute excessive amounts of the concentrates to the final working concentration Prepare only as much as you need for each assay Store PVDF membranes at ambient temperature in a sealed bag protected from light and moisture Store background quenching sheets in a sealed bag at ambient temperature D 0501 4 012 3 Additional Materials Required e Primary antibodies mouse and or rabbit IgG e Electrophoresis apparatus power supply gels and buffers for standard Laemmli SDS PAGE e Electro blotting apparatus and transfer buffer see Reference 4 e Methanol e 1x PBS or TBS without Tween these buffers are available from Advansta product numbers R 01038 020 and R 01039 020 respectively e Forceps with flat smooth tips Plastic non metal forceps are strongly recommended e Powder free gloves compatible with fluorescent applications Polyethylene gloves are strongly recommended e Incubation and washing trays with smooth interiors such as those provided by Advansta product numbers L 07031 L 07032 and L 07033 e Rotary or rocking platform a rocking platform is
18. preferred as mixing with a rocking action typically generates more uniform background than an orbital shaking e Fluorescent imager compatible with dyes excitable in green 530nm 560nm and red 600nm 650nm light Further details are discussed in the Imaging section www advansta com page 3 page 4 WesternBright MCF 4 Introduction to Multi color Fluorescent Western Blotting Western blotting provides a means to assay the presence and the expression level of a protein of interest in a complex mixture Proteins are separated electrophoretically transferred to a membrane substrate and the protein of interest is detected with specific antibodies 1 2 The antibody specific to the protein of interest can be directly labeled for example using radioactivity or can be detected by the use of a labeled secondary antibody Frequently a secondary antibody conjugated to horseradish peroxidase HRP is used and the location of the protein is detected via chemiluminescence 3 When using chemiluminescence only one protein can be detected per blot Assaying a second protein requires stripping and re probing the blot a time consuming procedure Imaging a Western blot using fluorescence allows for multiple proteins to be assayed on one blot by using secondary antibodies labeled with fluorophores having unique excitation and emission spectra 3 The WesternBright MCF kit provides the means to assay two proteins on a single
19. t Western Section Page blotting kit catalog number K 12021 010 Please see the Kit Contents section for details 1 Kit Contents 3 2 Shipping and Storage Conditions 3 3 Additional Materials Required 4 Plarage Informatiu 4 Introduction to Multi color Fluorescent Western Blotting 5 Unused kits can be stored at 4 C before opening After opening some components of 5 Overview of the Protocol for Fluorescent Western Blots 7 the kit may be stored at room temperature Please see the labels on each component and l refer to the Shipping and Storage section of this manual for details Do NOT freeze below 6 Quick Protocol 8 0 C 7 Detailed Protocol 9 8 Imaging 12 9 Troubleshooting and FAQ 12 License and Pafety Informatiu 10 References 14 The purchase of the WesternBright MCF Fluorescent Western blotting kit includes a limited 11 Related Products 14 cense to use it for life science research applications only MO other ICenISes are granen 12 Warranty badkcover Materials and or components of the kit may not be re sold or included into any commercial product offered for sale without prior permission Please contact us if the product is intended for commercial use or if you wish to obtain re sale or distribution rights Warning For research use only Not for clinical use Not for internal use in animals or humans Not for diagnostic use Not for household or any other unintended use Wear protective clothing such as protective glasses glo
20. ves and appropriate laboratory coveralls Avoid contact with skin or eyes In case of contact with skin or eyes flush immediately with large amount of water Refer to appropriate MSDS or safety statement document for more information Warning All solutions included in the kit contain 1 ug ml pentachlorophenol as a preservative against bacterial growth Pentachlorophenol is a hazardous material However at 1 ug ml it does not require any special handling beyond standard laboratory safety practices When diluted to final working concentrations as directed in the Protocol it no longer provides an anti bacterial protection Prepare only as much of each reagent as necessary to complete your current experiment D 05014 012 www advansta com page 1 page 2 WesternBright MCF 1 Kit Contents The WesternBright fluorescent Western blotting kit contains sufficient materials and supplies to perform ten dual color fluorescent Western blotting experiments when using a typical 8x10 cm sized polyacrylamide mini gel The following components are included R 05051 050 APC goat anti rabbit IgG antibody 50 ul R 05052 050 RPE goat anti mouse IgG antibody 50 ul L 08001 010 Low fluorescence PVDF transfer membrane 10 each 9x7 cm L 07001 010 Background Quenching Sheets 10 each R 03023 C50 AdvanBlock PF protein free blocking solution 5x concentrate 50 ml R 03024 D12 AdvanWash immunoblot washing solution 10x concentrate 120 ml 2 Phi
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