lot Detection with Radiance Substrate: A Detailed Protocol

4 PROTOCOLS 4.1. HRP/Chemiluminescent Blot Detection with Radiance substrate Long Protocol for Catalog Numbers • AC2100—Radiance Sample, sufficient for 140 cm2 membrane • AC2101—Radiance, 150 ml, sufficient for 1500 cm2 membrane Additional Materials Required • Electrophoresis apparatus and buffers for SDS-PAGE • Tank and buffers for electrophoretic transfer of proteins from gel to membrane •  Nitrocellulose or PVDF membrane, cut to size of gel. All membrane products available from Azure (see the Related Products section) are compatible with Radiance. • Washing buffer (PBS-T or TBS-T). For best results, use Azure Fluorescent Blot Washing Buffer (see the Related Products section) • Blocking buffer • Primary antibody compatible with your application • Secondary antibody, conjugated to Horseradish peroxidase (HRP) corresponding to your primary antibody (see the Related Products section) • CCD-based detection system, or film (see the Related Products section) Background Radiance chemiluminescent horseradish peroxidase (HRP) substrate is specially formulated for CCD imaging and to provide excellent performance with Azure’s cSeries imaging systems. Radiance produces a strong, long-lasting signal, which, combined with very low background levels, allows for long exposure times enabling the detection of lowabundance proteins. Additionally, the signal from Radiance is linear with respect to protein amount over a broad range of concentrations, displaying no substrate depletion at high protein loads, allowing the user to take full advantage of the linear range of the CCD detection method. Radiance is also compatible with X-ray film detection, though the limited dynamic range of film will make resulting data less quantitative. Since 1988, enhanced chemiluminescence or ECL (1) has become one of the most common detection methods in Western blotting (2). In this method, the secondary antibody is conjugated to the enzyme Horseradish peroxidase (1,2). Once bound to the membrane, the secondary antibody is detected by incubating the blot with a solution containing an HRP substrate that generates a light-emitting product after reaction with HRP (Figures 4.1, 4.2). The chemiluminescent signal can be detected by exposing the blot to X-ray film, or by imaging with a CCD camera. Radiance is an enhanced chemiluminescent substrate specially developed for CCD imaging. Radiance produces a bright signal with very low background for extremely high sensitivity and a detection limit of attomoles of protein. Additionally, the Radiance signal is long lasting, which combined with the low background, allows long-term exposures to detect low-abundance proteins. Substrate Light Oxidized Products HRP Secondary Antibody HRP Conjugate NH₂ NH₂ O NH Primary Antibody O O- HRP + H₂O₂ NH O- Blocker Membrane O Proteins transferred to the membrane Figure 4.1. The principle of chemiluminescent Western blotting. O Luminol Figure 4.2. Chemiluminescence of luminol. + N₂ + light Overview of the Protocol for Chemiluminescent Western Blots Electrophoresis to separate proteins in sample Transfer proteins from gel to membrane Block to mask nonspecific protein binding sites on membrane Primary antibody binds to protein of interest Wash to remove excess antibody Secondary antibody binds to primary antibody Wash to remove excess antibody Substrate (Radiance) substrate reacts with HRP bound to secondary antibody to create luminescent signal Image detect luminescent signal with CCD camera or film Quick Protocol For additional information, see the detailed protocol which follows. Step 1. Prepare your protein blot 2. Block membrane for 1 hour at room temperature (RT) 3. Incubate blot with primary antibody for one hour at RT with gentle agitation 4. Wash blot: • 1 x quickly • 1 x 15 min, with 1 ml/cm2 membrane • 3 x 5 min, with at least 0.5 ml/cm2 membrane each time 5. Incubate blot with secondary antibody for one hour at RT with gentle agitation 6. Wash blot: • 3 x 5 min, with at least 0.5 ml/cm2 membrane each time 7. Mix Radiance components 1:1 and place 0.1 ml/cm2 on blot for 2 minutes 8. Drain excess reagent 9. C  over damp blot with plastic wrap and image by CCD camera or exposure to X-ray film User Notes Detailed Protocol Step Notes 1. Prepare a protein blot 1.1. Separate the protein sample(s) via electrophoresis 1.2. Transfer proteins to membrane • Prewet membrane in transfer buffer, and assemble transfer sandwich according to tank manufacturer’s instructions. • Dot-blots or slot blots can also be detected with Radiance.  ny electrophoresis system and buffer, such as Laemmli, is compatible with Radiance. • A  wet or tank transfer method is preferred, though semi-dry methods should also • A be compatible. We have found that the buffer system developed by Bolt et all (3) works well.  oth nitrocellulose and PVDF membranes are compatible with Radiance. • B • If using PVDF, first wet membrane with a 1 min incubation in 100% MeOH followed by water for ~5 min and then transfer buffer for 5-10 min.  slot blot applications, nitrocellulose is much more convenient than PVDF because • For it is more difficult to avoid bubbles with PVDF. 2. Block membrane • Incubate the blot in a blocking buffer with gentle agitation for 1 hour at room temperature (RT). Use 0.2 to 0.5 ml of blocking buffer per cm2 of blot to provide adequate blocking. • Blocking masks non-specific protein binding sites on the membrane, reducing background and increasing the specificity of binding of the primary antibody to the protein of interest. • The optimal blocking buffer will depend in part on the nature of the antigen of interest, and on the quality of the primary antibody. Common blocking agents including nonfat dry milk have been found to be compatible with Radiance. • 10 to 20 ml is usually sufficient for a typical 7 x 9 cm mini-blot. 3. Incubate blot with primary antibody • Dilute primary antibody in blocking buffer. • Incubate blot with primary antibody solution for 1 hour at RT with gentle agitation.  • Optimal primary antibody dilutions must be determined empirically. • For CCD imaging, we recommend primary antibody dilutions from 1:1000 to 1:10,000. A good initial dilution is 1:5000. • If the blot will be imaged on film, use 2–5x less primary antibody than for CCD imaging. For example, if 1:1000 dilution of the primary antibody was optimal for CCD detection, 1:5000 is suitable for film detection. • A  ntibody can be added to a dish and placed on a shaker, or a smaller volume (5-10 ml) can be used by sealing the blot into a bag and placing it on a rotary platform. 4. Wash blot to remove excess primary antibody • 1 x quickly • 1 x 15 min, with 0.7 ml/cm membrane • 3 x 5 min, with at least 0.3 ml/cm2 membrane each time. • For  best results, use Azure Fluorescent Blot Washing Buffer (AC2113) which is optimized for chemiluminescent as well as fluorescent blots. PBS-T or TBS-T are also compatible with Radiance. • W  e recommend washing or rocking blots in a clean dish on a shaker to provide gentle agitation. • F  or example, a standard 7x9 membrane requires: ~50 ml of washing solution for the 15 min wash; and ~20 ml of washing solution for 5 min washes. 5. Incubate blot with secondary antibody • Dilute secondary antibody in blocking buffer. • Incubate blot with secondary antibody solution for 1 hour at RT with gentle agitation. • Optimal  secondary antibody dilutions must be determined empirically. • W  e recommend secondary antibody dilutions of 1:5,000 to 1:20,000. A good initial dilution is 1:10,000. • If  the blot will be imaged on film, use 2–5x less secondary antibody than for CCD imaging. 1:50,000 dilution is a good starting point for film detection. • S  ee also notes for step 3. 6. Wash blot to remove excess secondary antibody • 3 x 5 min, with at least 0.3 ml/cm2 membrane each time. • See  notes for step 4. 7. Incubate blot with Radiance • Mix  components 1 and 2 in a 1:1 ratio in sufficient amounts to obtain at least 0.1 ml/cm2 of the blot and add to the blot. • It is better to prepare the working solution just before use. However, mixed reagent is stable for several hours at RT. • Allow substrate to react with blot for 2 minutes. • Be careful not to touch or put pressure on the blot as this can result in non-specific background. • Use only plastic forceps, not metal; metal forceps damage the blocked surface, creating new adsorption sites. Also, traces of metal may act as a catalyst for nonenzymatic substrate oxidation, resulting in very high background. • The minimal amount of working reagent is 0.1 ml/cm2. For example, for a 7 x 9 cm blot, this minimal volume is 7x9x0.1=6.3 ml. • If using the minimal amount of working reagent, incubation may be done without agitation. Make sure the membrane surface is level so adequate reagent is held by surface tension. • Incubation may also be done with gentle agitation in a tray just slightly larger than the membrane. Increase the reagent volume as necessary to ensure the membrane is adequately covered with reagent. 8. Drain excess reagent • Remove  excess substrate via capillary action by touching a KimWipe® or other absorbent material to the edge of the blot. 9. Image blot • While blot is damp, cover with transparent plastic wrap and either place blot in CCD imager, or expose blot to film. • We  recommend trying three exposures; 30 sec, 2 min, and 5 min. • The blot can be imaged and re-imaged for several hours; 70% of the initial signal will remain after 60 minutes, and substantial signal will remain after 8-10 hours. Troubleshooting & 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: Problem Possible Soutions High background • • • • No or low signal • Check that correct primary antibody used. • Check that secondary antibody recognizes primary (for example if the primary is a rabbit antibody, that the secondary is goat-anti-rabbit). White spots within bands • Improve transfer, making sure to remove any bubbles between the gel and the membrane. Speckled background • Filter secondary antibody. • Filter blocking and washing buffers. • Ensure that the laboratory environment is clean, to minimize dust, debris or any other particles that might come in contact with the blot. Cover the dish during incubation or washing steps. • Use non-powdered gloves, or switch to a different kind of gloves. We recommend powderfree nitrile gloves or polyethylene gloves. Reduce primary antibody concentration by increasing the dilution factor. Try a different blocking buffer. Try a shorter exposure time. Increase washing time. References 1.  Thorpe GH, Kricka LJ, Moseley SB, Whitehead TP, Phenols as enhancers of the chemiluminescent horseradish peroxidaseluminolhydrogen peroxide reaction: application in luminescencemonitored enzyme immunoassays. Clin Chem. 1985 Aug; 31(8): 1335-41. 2. Leong MM, Fox GR., Enhancement of luminol-based immunodot and Western blotting assays by iodophenol. Anal Biochem. 1988 Jul; 172(1): 145-50. 3.  Bolt M.W., Mahoney P.A, High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electophoresis. Anal Biochem. 1997 May 1; 247(2): 185-192. Related Products Catalog Number Product Size AC2113 Azure Fluorescent Blot Washing Buffer 500 ml AC2114 Goat-anti-rabbit HRP-conjugated secondary antibody 500 μl AC2115 Goat-anti-mouse HRP-conjugated secondary antibody 500 μl AC2105 Low Fluorescence Western Membrane (PVDF) 7x9 cm 10 sheets AC2106 Nitrocellulose Transfer Membrane 0.45 μm 7x9 cm 10 sheets AC2107 Nitrocellulose Transfer Membrane 0.22 μm 7x9 cm 10 sheets 4.2. Fluorescent Western Blot Protocol with AzureSpectra Reagents Quantitative, multi-color and near-IR fluorescent Western blotting kits. Long Protocol for Catalog Numbers • AC2193—AzureSpectra rb650/ms550 Kit • AC2191—AzureSpectra IR rb700/ms800 Kit • AC2192—AzureSpectra IR ms700/rb800 Kit All kits • Low-fluorescence PVDF transfer membrane 9x7 cm. 10 each • Background Quenching Sheets, 10 each • Azure Fluorescent Blot Blocking Buffer, 1x ready-to-use solution, 300 ml • Azure Fluorescent Blot Washing Buffer, 10x concentrate, 250 ml For AC2193 • Goat-anti-rabbit IgG 650, 40 μl • Goat-anti-mouse IgG 550, 40 μl For AC2191 • Goat-anti-rabbit IgG IR700, 40 μl • Goat-anti-mouse IgG IR800, 40 μl For AC2192 • Goat-anti-mouse IgG IR700, 40 μl • Goat-anti-rabbit IgG IR800, 40 μl Shipping and Storage Conditions Before opening, the kit may be stored at +4°C. AzureSpectra fluorescent secondary antibody conjugates may be stored at +4˚C for up to a month. For longer term storage, store antibody conjugates at -20˚C. Gently mix and briefly spin the tubes before taking aliquots.  Azure Fluorescent Blot Blocking Buffer must be stored at +4˚C. Azure Fluorescent Blot Washing 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. Additional Materials Required • Primary antibodies: mouse and/or rabbit IgG. • Electrophoresis apparatus, power supply, gels and buffers for standard Laemmli SDS-PAGE. • Electro-blotting apparatus and transfer buffer (see Reference 4). • Methanol. • 1x PBS or TBS without Tween. • Forceps with flat smooth tips. Plastic (non-metal) forceps are strongly recommended. • Powder-free gloves compatible with fluorescent applications. Polyethylene gloves are strongly recommended. •  Incubation and washing trays with smooth interiors such as those provided by Azure (product numbers AC2120, AC2123, AC2150 and AC2153). •  Rotary or rocking platform – a rocking platform is preferred as mixing with a rocking action typically generates more uniform background than orbital shaking. • AzureSpectra rb650/ms550 Kit (AC2193): Fluorescent imager compatible with dyes excitable in green (530nm – 560nm) and red (600nm – 650nm) light. Further details are discussed in the “Imaging” section. • A   zureSpectra IR rb700/ms800 and ms700/rb800 Kits (AC2191 and AC2192): an imager compatible with dyes excitable in far-red (630nm – 700nm) and near infrared (750nm – 780nm) light. Further details are discussed in the “Imaging” section. Until recently fluorescent reporters were not widely used for Western blotting applications for various reasons. Membranes typically used for Western blotting had high levels of autofluorescence, appropriate imaging instruments were not readily available, and the time required to acquire an image of a typical blot was relatively long. The most important factor that prevented the use of some fluorescent detector molecules in Western blotting applications was that these proteins need to remain hydrated to maintain their high levels of fluorescence. Western blot membranes can dry quickly and lose the water necessary to sustain fluorescence, and, unfortunately, re-hydrating the membrane does not restore the fluorescence of certain detector fluorophores. Today, all the above problems can be easily addressed. PVDF membranes with low autofluorescence have become available, and there are several choices of fast and high resolution fluorescent imaging instruments including laserbased scanners and LED-based gel documentation imagers. The most difficult problem is to preserve the hydration of fluorescent detector molecules long enough to be able to image the blot within a reasonable period of time. We have developed the Azure Protein Free Blocking Buffer and Azure Fluorescent Blot Blocking Buffer to address this problem. The blocking solution provided with the kit contains a component that provides an efficient blocking of the PVDF membrane from non-specific protein binding. At the same time, Absorbence/Intensity The AzureSpectra rb650/ms550 Kit and AzureSpectra IR rb700/ms800 and ms700/rb800 Kits provide the means to assay two proteins on a single Western blot. Each kit contains two secondary antibody conjugates, each pre-labeled with a different fluorescent reporter. Fluorescent reporters utilized in the AzureSpectra rb650/ms550 Kit are excitable in the visible fluorescence range. The excitation and emission spectra of of the AzureSpectra 550 and 650 conjugates are shown in Figures 4.3 and 4.4. Fluorescent reporters used in the AzureSpectra rb700/ms800 and ms700/rb800 Kits are excitable by far-red and near-infrared light. The excitation and emission spectra of the AzureSpectra NIR conjugates are show in Figures 4.5 and 4.6. 515 nm ex 565 nm em 300 350 400 450 500 550 Wavelength (nm) 600 650 700 Figure 4.3. Absorption and emission spectra of AzureSpectra 550 goatanti-mouse IgG antibody conjugate (AC2159) 692 nm ex 709 nm em 250 300 350 400 450 500 550 600 650 700 750 800 850 Wavelength (nm) Figure 4.4. Absorption and emission spectra of AzureSpectra 700 IgG antibody conjugate (AC2128 and AC2129) Absorbence/Intensity  When using chemiluminescence, only one protein can be detected per blot. Assaying for 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). 250 250 653 nm ex 672 nm em 300 350 400 450 500 550 600 Wavelength (nm) 650 700 750 800 Figure 4.5. Absorption and emission spectra of AzureSpectra 650 goatanti-rabbit IgG antibody conjugate (AC2165) Absorbence/Intensity 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). Absorbence/Intensity Introduction to Multi-Color Fluorescent Western Blotting 783 nm ex 800 nm em 250 300 350 400 450 500 550 600 650 700 750 800 850 Wavelength (nm) Figure 4.6. Absorption and emission spectra of AzureSpectra 800 IgG antibody conjugate (AC2135 and AC2136) 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 a protein of interest. When using high quality primary antibodies and optimized assay conditions, single picogram detection can routinely be achieved. The secondary antibody conjugates provided in the AzureSpectra rb650/ ms550 Western Kit and AzureSpectra IR rb700/ms800 and ms700/rb800 Kits are labeled anti-mouse IgG and antirabbit IgG conjugates. Therefore, the AzureSpectra kits are appropriate for experiments incorporating most readily available primary antibodies, raised in mouse and/or rabbit. A schematic representation of the twocolor Western blot is shown in Fig. 4.7. DYE 1 DYE 2 Secondary Antibody Conjugate Secondary Antibody Conjugate Primary Antibody (mouse) Primary Antibody (rabbit) Blocking Compound PVDF Membrane Proteins transferred to the membrane Figure 4.7. Schematic diagram of the two-color assay utilized in the AzureSpectra Western Kits. 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 Azure Fluorescent Blot Blocking Buffer Incubate with primary antibodies dilute antibodies in 1x Azure Fluorescent Blot Blocking Buffer, incubate for 1 hour Wash the membrane with 1x Azure Fluorescent Blot Washing Buffer • 2 x 30 sec – 1 min (rinse); • 3 x 5 min Incubate with secondary antibodies dilute antibodies in 1x Azure Fluorescent Blot Blocking Buffer, incubate for 1 hour Wash the membrane in 1x Azure Fluorescent Blot Washing Buffer • 2 x 30 sec – 1 min (rinse); • 3 x 5 min Rinse the membrane in PBS or TBS without Tween-20 5 min Place the membrane on a background quenching sheet and image 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: • 2 x quickly • 3 x 5 min with 25 ml each time. 5. Incubate blot with secondary antibody for one hour at RT with gentle agitation. 6. Wash blot with 1x washing buffer: • 3 x 5 min with 25 ml wash buffer each time • 1 x 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. Detailed Protocol Step Notes 1. Protein separation and transfer 1.1. Separate protein samples by polyacrylamide gel electrophoresis using a mini-sized gel (i.e. 8 x 10 cm). 1.2. 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. 1.3. Pre-wet the membrane in methanol for a few moments until the membrane is completely wet. 1.4. T  ransfer 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. 1.5. Transfer the membrane from water to transfer buffer, and incubate for 5 minutes. 1.6. Assemble the transfer sandwich as required for your transfer apparatus. 1.7 Perform the transfer overnight at 15 V, or for 1 to 2 hours at 70 V with an ice pack. • A  semi-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. • F  or a fast transfer method that works well for fluorescent blots, we recommend Azure Transfer Buffer (available from Azure, product number AC2127). When using Azure Transfer Buffer, the transfer should be carried out for 15 to 30 minutes at 12 V/cm. 2. Membrane blocking and binding of the primary antibodies 2.1. Once the transfer is completed (step 1.7), remove the membrane from the sandwich and place it into a tray with Milli-Q or distilled water for 5 minutes. 2.2. Transfer 10 ml of 1x Azure Fluorescent Blot Blocking Buffer to an incubation tray. Make sure that the tray is of an appropriate size for your membrane. 2.3. Transfer the membrane from water into Azure Fluorescent Blot Blocking Buffer and block the membrane for 10 minutes with gentle agitation. 2.4. While the membrane is in the Azure Fluorescent Blot Blocking Buffer, prepare the incubation mixture containing your primary antibody or an appropriate mixture of primary antibodies diluted in 10 ml 1 x Azure Fluorescent Blot Blocking Buffer. Gently mix the incubation solution by inverting the tube several times. Do NOT vortex. 2.5. After the blocking is completed (step 2.3), remove the solution from the tray and add the solution of primary antibodies to the membrane. 2.6 Incubate the membrane with the solution of primary antibodies for 1 hour at room temperature with gentle agitation. • 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 orbitalshaker mixing. • Incubation of the membrane 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. • 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. 3. Washing the membrane 3.1. About 10 minutes before the end of the incubation step, prepare 300 ml of 1x Azure Fluorescent Blot Washing Buffer. Mix 30 ml of the Azure Fluorescent Blot Washing Buffer concentrate provided in the kit with 270 ml of purified water (Milli-Q quality or distilled). This amount is sufficient to complete the experiment. 3.2. Add 25 ml of the Azure Fluorescent Blot Washing Buffer to a new tray.  fter the incubation (step 2.6) is completed, transfer the 3.3. A membrane from the incubation tray into the tray with the Azure Fluorescent Blot Washing Buffer prepared in step 3.2. Use clean forceps to transfer the membrane. Make sure that the orientation notch is in the same position so that the side of the membrane containing the transferred proteins is facing up. 3.4. R  inse the membrane twice for 30 seconds to 1 minute each time with gentle agitation. 3.5. Remove the Azure Fluorescent Blot Washing Buffer completely or transfer the membrane into a new clean tray. 3.6. Wash the membrane 3 more times for 5 minutes each with gentle agitation, replacing with 25 ml of fresh Azure Fluorescent Blot Washing Buffer each time. • 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. 4. Probing the membrane with the AzureSpectra secondary antibody fluorescent conjugates 4.1. During the last washing step, prepare the mixture of secondary antibody fluorescent conjugates. Transfer 10 ml of the Azure Fluorescent Blot Blocking Buffer into a new 15 ml tube. Add 4 μl each of the labeled conjugates provided in the kit (dilution factor 1:2500). If using an AzureSpectra NIR Kit, 1 μl each of the IR700- and IR800-labeled conjugates provided in the kit (dilution factor 1:10,000) may be used. 4.2. A fter the last washing step is completed, remove the Azure Fluorescent Blot Washing Buffer 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. • When  diluting the secondary antibody conjugates, gently mix by inverting the tube several times. Do NOT vortex. 5. Final washing 5.1. Remove the incubation solution and wash the membrane as in step 3.4–3.7 using Azure Fluorescent Blot Washing Buffer with gentle agitation. 5.2. R  emove the Azure Fluorescent Blot Washing Buffer and add 20-50 ml of PBS or TBS that does NOT contain Tween-20 or any other detergents and wash the membrane for 5 minutes with gentle agitation. 5.3. R  emove the membrane from the solution with clean forceps and transfer onto a black background quenching sheet and allow the excess liquid to run off the membrane by holding the plastic sheet with the membrane vertically. • The  purpose of step 5.2 is to remove from the membrane Tween-20 that is present in Azure Fluorescent Blot Washing Buffer. Removal of Tween-20 stabilizes the structure of the AzureSpectra conjugates and preserves the fluorescence when the membrane dries. • T  he membrane will not fall off the plastic sheet as it adheres to it while still wet. Do not use any wipes or tissue to remove the excess liquid as this may increase the background and introduce random fluorescent artifacts. Minor liquid excess does not affect the results. Imaging  The membrane can be imaged immediately after the final wash step. For blots conducted with AzureSpectra rb650/ ms550 Western Kit, the highest sensitivity is achieved when the membrane is dry. 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. Blots conducted with AzureSpectra IR rb700/ms800 and ms700/rb800 Kits can be imaged wet or dry. Review the absorption and emission spectra of the AzureSpectra conjugates included in your kit (Fig. 1 through 4) to choose appropriate setting of your imaging instrument. In most cases the settings appropriate for imaging Cy3 and Cy5 fluorescent dyes will work well for imaging the AzureSpectra 550 and 650 conjugates, and settings appropriate for imaging IR680 and IR800 fluorescent dyes will work well for the AzureSpectra IR700 and IR800 conjugates respectively. Troubleshooting & 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 recommendations for primary antibody dilution? Follow the manufacturer’s recommendations for the antibody dilution. Typical antibody dilutions for primary antibodies range from 1:250 to 1:5000. High concentrations of primary and secondary antibodies can result in high background levels on the blot. Can we use nitrocellulose membranes? Nitrocellulose membranes have very high autofluorescence that makes it impossible or very difficult to detect low amounts 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. IPFL00010). What kinds of transfer methods are acceptable for use with the kit? We currently suggest using a standard tank transfer method for fluorescent Western blotting applications. Semi-dry transfer systems do not work well for fluorescent applications as they typically generate relatively high and non-uniform background, especially in the channel excited with green light. Azure’s Azure Transfer Buffer can be used for a quick wet transfer. Can we dry the blot and image [n] days after we do the Western assay? The best performance for the blot is attained within 2 hours of drying it. In order to preserve the fluorescence on the dried blot when using AzureSpectra rb650/ms550 Western Kit, it is important that Tween-20 is completely removed. See step 5.2 for details. If necessary, step 5.2 may be repeated one more time. The membrane can be dried after this point. However, lower protein signal levels are expected with prolonged storage. Can we use the blocking solution and/or wash solution we typically use with our current blotting system? We advise against using alternate blocking or washing solutions. Alternate components may result in a higher background or reduced sensitivity. When the membrane is dried after using other blocking solutions, the fluorescent intensity and stability of fluorophores may be reduced to a greater extent. If you intend to use your blocker, we suggest using using Azure Protein Free Blocking Buffer or Azure Fluorescent Blot Blocking Buffer for membrane blocking (step 2.4), and using your specific blocker to prepare the primary antibody incubation solution (step 2.5). We do not detect signal on the blot. Check to see if the transfer was successful by using a protein standard; if this is positive, check the imaging system to confirm the correct excitation and emission settings. Typically, imaging channels optimized for Cy3 and Cy5 dyes work well with the AzureSpectra 550 and 650 conjugates and settings for IR680 and IR800 dyes for AzureSpectra 700 and 800 conjugates. If you are trying to detect small amounts of a target protein, try to increase the concentration of your primary antibody first. If this is unsuccessful, also increase the concentration of the secondary conjugates. Our lab generally uses Tris/ Phosphate based buffers; will this work with the kit? We suggest using the Blocking and Washing solutions supplied with the kit to guarantee best performance. However if you require different buffer conditions, test a small blot before using larger quantities of kit components. However, if you do not use the Azure Protein Free Blocking Buffer or Azure Fluorescent Blot Blocking Buffer reagent, you may lose the benefits of its protective effect on the fluorescence stability when your membrane dries. References 1. Towbin  H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979: 76 (9): 4350-4354. 2. Burnette  WN. “’Western blotting’”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981; 112(2):195203. 3. Patton,  Wayne F. A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis. 2000; 21: 1123-1144. 4.3. Azure HRP Stripping Buffer Stripping buffer for chemiluminescent Western blots. Short Protocol for Catalog Number • AC2154—HRP Stripping Buffer Storage Information Azure HRP Stripping Buffer is stable at room temperature (4°C-25°C) for at least one year. Warnings and Precautions • Azure HRP Stripping Buffer is for research use only. • Always wear gloves when handling membranes and reagents. • Refer to MSDS for additional safety information. • The product is guaranteed to be free of manufacturer defects, and to function as described when the provided protocol is followed by properly trained personnel. Short Protocol Step User Notes 1. Rinse blot with 20 mL water at room temperature for 5 minutes with agitation. 2. Strip blot using 20 mL HRP Stripping Buffer at room temperature for 5 minutes with agitation. Ensure blot is completely covered in stripping buffer. 3. Wash blot with 1x PBST or 1x TBST for 5 minutes with agitation. Repeat twice for a total of 3 washes. 4. Block membrane and perform antibody incubations. Tips • Azure HRP Stripping Buffer is compatible with both nitrocellulose and PVDF membranes. • Because stripping blots may reduce signal intensity, probe for the lowest expressed antigen first, for best results. 4.4. AzureRed Fluorescent Protein Stain Fluorescent total protein stain for gels and blots. Protocol for Catalog Number • AC2124—AzureRed Fluorescent Protein Stain Storage Information  Store AzureRed Dye in a freezer at -15 ºC to -30 ºC in the original brown bottle provided and protect from light. The AzureRed Powder A and Powder B are stable at room temperature for one year. Warnings and Precautions • AzureRed total protein stain is for research use only. • Always wear gloves when handling membranes and reagents. • Refer to MSDS for additional safety information. • 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. Kit Contents (AC2124) AzureRed total protein stain, 5 ml • AzureRed Powder A 4 packets, 10.1 g each • AzureRed Powder B 23.4 g • AzureRed Dye 5 ml AzureRed Stain, 5 ml, kit is sufficient for staining twenty SDS-PAGE mini-gels (8 cm x 11 cm), four fill-sized 2D gels (17 cm x 17 cm), or forty mini-blots (9 cm x 7 cm). Shipping and Storage Conditions Product may be shipped refrigerated or frozen on blue ice or dry ice. Shipping at ambient temperature (below 27°C) is acceptable if the total dispatch time is no longer than 5 days. Upon receipt, store AzureRed Dye in a freezer at -15 ºC to -30 ºC in the original brown bottle provided and protect from light. AzureRed Powder A and Powder B may be stored at room temperature in a dry location. Additional Materials Required • High-purity water (distilled, Milli Q, or equivalent) • 100% ethanol • Staining tray • Shaking or rocking platform About AzureRed AzureRed is based on epicocconone, a small, naturally occurring fluorescent compound1 that reversibly binds to lysine, arginine, and histidine residues in proteins and peptides to yield an intensely redfluorescent product.2 This unique mechanism provides sensitive quantification of proteins in 1D and 2D gels of all chemistries, on both PVDF and nitrocellulose blots3-5 and provides unparalleled compatibility with Mass Spectrometry.6-8 Normalized Excitation Normalized Emission Excitation and Emission Spectra The excitation and emission spectra of AzureRed can be seen in Figure 4.8. Figure 4.8. Excitation and Emission Spectra of AzureRed Dye. Overview of AzureRed Membrane Staining Protocol Rinse Rinse membrane in ultra pure water for 5 minutes Staining Incubate membrane in Stain Solution for 15 to 30 minutes Fixation Incubate membrane in Fix Solution for 5 minutes Washing Wash membrane in methanol 2 times for 3 minutes each Image or Proceed with Western Blotting Preparation of Solutions  staining, prepare Fix, Stain, and Wash solutions as described below. These solutions are stable for up to 1 Before 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 AzureRed 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 packet. •  Solution—Add contents of one AzureRed Powder A packet (10.1 g) to 850 ml of high-purity water in a 1 L Fix bottle. Mix until dissolved. Add 150 ml 100% ethanol and mix thoroughly. • Stain Buffer—Add contents of one AzureRed Powder B packet (23.4 g) to 1 L 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.  Solution Gel Dimensions Fix Stain Stain Buffer AzureRed Dye Wash Fix 8 cm x 11 cm x 1 mm (mini-gels) 100 ml 50 ml 250 μL 100 ml 100 ml  3.3 cm x 8.7 cm x 1 mm 1 (small format 2D gels) 200 ml 100 ml 500 μL 200 ml 200 ml 17 cm x 17 cm x 1 mm 500 ml 250 ml 1.25 ml 500 ml 500 ml 17 cm x 17 cm x 1.5 mm 500 ml 250 ml 1.25 ml 500 ml 500 ml 15 cm x 19 cm x 1 mm 500 ml 250 ml 1.25 ml 500 ml 500 ml 15 cm x 19 cm x 1.5 mm 500 ml 250 ml 1.25 ml 500 ml 500 ml 20 cm x 25 cm x 1 mm 750 ml 375 ml 1.875 ml 750 ml 750 ml 20 cm x 25 cm x 1.5 mm 750 ml 375 ml 1.875 ml 750 ml 750 ml Table 4.1. Volumes of Solutions For Different Gel Sizes. Azure Imager Recommended Imaging Channel c150, c200, c280, c300, c500 UV365 c400, c600 Cy3 Sapphire Biomolecular Imager – RGB, RGBNIR 520 Table 4.2. AzureRed is imageable with both UV and green light. However, best sensitivity is achieved when detected with green light. Detailed Protocol, Blot Staining Step Notes 1. Washing • Following transfer, wash blot for 5 min in water. • Proceed to PVDF (2) or Nitrocellulose (3) protocol. • F  or 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. • For all steps, use 50 ml for small blots, 400 ml for large. 2. PVDF Protocol 2a. Staining • Place blot protein side down in Stain Solution. • Stain blot with gentle rocking for 15–30 min. • Prepare  Stain Solution: Allow AzureRed Dye to warm to room temperature. Mix thoroughly. For small blots, dilute 125 μl AzureRed Dye in 50 ml Stain Buffer. Mix well. • F  or large blots, dilute 1 ml of AzureRed Dye in 400 ml Stain Buffer. Mix well. 2b. Acidification • Place blot in Fix Solution and incubate with gentle rocking for 5 min. • Blot will appear green. 2c. Wash • Rinse blot 3 times with 100% ethanol for 2–3 min each, until green background on blot has been completely removed. • Methanol may used instead of ethanol. 2d. Drying • Hang blot from a peg or dry on wire mesh to allow blot to dry evenly. • Allow blot to dry completely before imaging. • If using in a multiplex Western blot, do not dry membrane after staining. Upon completion of the washing step, proceed directly to blocking the membrane. 3. Nitrocellulose Protocol 3a. Staining • Place blot protein side down in Stain Solution. • Stain blot with gentle rocking for 15–30 min. 3b. Acidification • Place blot in Fix Solution and incubate with gentle rocking for 5 min. 3c. Washing • Wash blot 1 time in Wash Solution for 5 min with gentle rocking. • Wash blot 2 times in high-purity water for 5 min with gentle rocking. • Prepare  Stain Solution: Allow AzureRed Dye to warm to room temperature. Mix thoroughly. For small blots, dilute 125 μl AzureRed Dye in 50 ml Stain Buffer. Mix well. • F  or large blots, dilute 1 ml of AzureRed Dye in 400 ml Stain Buffer. Mix well. • Blot will appear green. 3d. Drying • Allow blot to dry completely before imaging. • If using in a multiplex Western blot, do not dry membrane after staining. Upon completion of the washing step, proceed directly to blocking the membrane. Detailed Protocol, Gel Staining Step Notes 1. Fixation • F  ix gel in Fix Solution for a minimum of 1 hr with gentle rocking. • For correct volumes at each step, refer to Table 4.1. • F  or gels thicker than 1 mm or backed gels, increase the fixation time to 1.5 hr. • The fixation time can be extended to overnight. 2. Staining • Prepare the Stain Solution immediately prior to staining. • Remove gel from Fix Solution and place in Stain Solution, minimizing carryover of the fixing solution. • Stain gel for 1 hour with gentle rocking. • To  prepare Stain Solution: Allow AzureRed Dye to warm to room temperature. Mix thoroughly, then dilute 1 part AzureRed Dye in 200 parts Stain Buffer. Mix well. Refer to Table 4.1 for volumes of solutions used for different gel sizes. • S  tain Solution will degrade over time. Prepare only as much as is needed and use immediately. • Increase staining time to 1.5 hours for gels 1.5 mm thick or backed gels. Extending the staining time to 2 hours will not affect results. • DO NOT stain for longer than 2 hours. 3. Washing • Remove gel from Stain Solution, rinse with high-purity water, and wash in Wash Solution for 30 min with gentle rocking. • For  1.5 mm thick gels, or gels with high background fluorescence, increase washing time to 45 min. 4. Acidification • Remove gel from Wash Solution and place in Fix Solution. • Incubate in Fix Solution for 30 min with gentle rocking. • This  step can be repeated or extended to overnight to reduce background staining. • If performing this step overnight, protect the gel from light. 5. Imaging • D  etect fluorescence at 610 nm using standard fluorescence scanners and CCD camera systems. For recommended imaging settings, refer to Table 4.2. • Compatible  excitation sources include green light and UV light. • D  etect fluorescence using a 610 nm band pass or 560 nm long pass filter. Destaining AzureRed 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. Storage  Gels may be stored at 4 ºC in 1% citric acid and protected from light. For extended storage (up to 6 months), add AzureRed 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.’  Troubleshooting & FAQ Problem Possible Soutions High background • Handle gels with clean non-powdered gloves, and avoid contamination with dust. • Ensure concentrated AzureRed Dye was brought to room temperature and thoroughly mixed prior to dilution. • Ensure stain was thoroughly mixed into Stain Buffer before adding to gel. • Stain only one gel per tray. • Use high-purity water (distilled, Milli-Q, or equivalent). No or low signal • 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. • Stain may fade with long exposure times and associated heating on CCD-based instruments. • Ensure stain concentrate was brought to room temperature and mixed thoroughly before dilution. • Staining for over 2 hours in alkaline conditions destabilizes proteins, and leads to diffusion of protein bands from the gel matrix. Negative staining • Use high-quality SDS in the preparation and running of the gel. • Extend fixation time to overnight. • Use correct volumes of Fix and Wash Solutions. • Extend washing time. Speckled background • Filter buffers to remove dust or precipitates. • Protect gel from airborne particles. 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. 2. Coghlan, D. R., Mackintosh, J. & Karuso, P. (2005). Mechanism of reversible fluorescent staining of protein with Epicocconone. Organic Letters. 7, 2401-240. 3.  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. 4.  Malmport, E., Mackintosh, J., Ji, H., Veal, D. & 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. 5.  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. & 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. 6. 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. 7. Nock, C.M., Ball, M. S., White, I. R., Skehel, 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. 8.  Ball, M. S., Karuso, P. (2007). Mass Spectral Compatibility of Four Proteomics Stains. Journal of Proteome Research. 6, 4313-4320.