Multiplex Western Blotting with AzureSpectra Antibodies

5 APPLICATION NOTES Product: cSeries | Application: Western blotting Three-Color Western Blots with AzureSpectra Antibodies Visualize Three Proteins at Once – with AzureSpectra labeled antibodies Classic western blotting techniques using chemiluminescent detection are typically useful for detecting one protein at a time. To evaluate multiple proteins of interest using chemiluminescence, stripping and re-probing the blot or splitting the samples into separate assays is usually required. Visualizing multiple proteins of interest within the same sample simultaneously – or multiplexing – is possible with fluorescent antibodies. Antibodies carrying different fluorophores (with non-overlapping spectra) can be detected simultaneously with the right imaging system. With a digital imager like the Azure c600 you can visualize three proteins at once. Why would you need to visualize multiple proteins at once? Convenience is an obvious benefit to multiplexing, as loading controls are often used for Western blot data. By using different fluorescently labeled secondary antibodies for the protein of interest along with the loading control, clear and quantifiable results are ready to be analyzed immediately, even if the proteins are close in molecular weight. Additionally, quantitation is increasingly desirable when publishing protein expression data. What if you have two different proteins of interest, such as phosphorylated and non-phosphorylated version of a protein? In order to include a loading control for accurate comparison of different lanes, a third color would be necessary. Conveniently, 3-color western blotting is possible and easy with AzureSpectra labeled antibodies and the Azure c600 system. The Azure c600 imaging system is designed for multicolor fluorescent Western blotting, with two nearinfrared (NIR) and 3 visible fluorescent channels. This application note demonstrates a procedure to obtain three color Western Blots using the Azure c600 and Azure’s broad selection of fluorescently labeled secondary antibodies. In addition to pre-labeled antibodies, Azure also offers an Antibody Labeling Kit that provides everything you need to fluorescently label your primary or secondary antibodies. The non-overlapping emission spectra of the fluorophores in each of the available dyes means you have the opportunity to easily perform multiplexing assays. Materials and Methods Run and transfer gel HeLa cell lysates were prepared and electrophoresed on a 4-15% Mini-Protean TGX gel, using 1, 2, 5, 10, and 20 µg of lysate for each lane, respectively. After electrophoresis and separation, proteins were transferred to a low fluorescence PVDF membrane using Azure transfer buffer. Western blotting After transfer, the membrane was blocked for 10 minutes then probed with 5 µg of mouse anti-GAPDH, 5 µg of rabbit anti-beta-actin, and 5 µg of rat anti-tubulin for 1 hour. The blot was rinsed twice and then washed three times for 5 min each with 25 mL of Azure IR wash buffer. Next, the blot was incubated for 1 hour with the labeled secondary antibodies: 2 µg of goat anti-mouse-550, 4 µg goat anti-rabbit-700, and 4 µg goat anti-rat-800. After incubation, the blot was washed as before in Azure IR wash buffer followed by a 5 min rinse in 25 mL of PBS. Step Materials Run Gel SDS Polyacrylamide Gel Transfer Block Blot Probe Blot Part number PVDF Membrane AC2105 Azure Transfer Buffer AC2127 AzureSpectra Fluorescent Blot Blocking Buffer AC2190 Primary Antibodies Specific for proteins of interest Azure IR Wash Buffer AC2145 Goat-anti-rat 800 AC2138 Goat-anti-rabbit 700 AC2128 Goat-anti-mouse 550 AC2159 PBS Table 1. Materials and product numbers. Image Western blot All 3 channels Tubulin (green channel) Beta actin (red channel) GAPDH (blue channel) After rinsing in PBS, the blot was allowed to dry before imaging on the Azure Biosystems c600 digital imager. Results and Conclusions The Azure c600 and AzureSpectra antibodies allow for quick and easy detection of three different proteins in the same samples on the same western blot. Accurate quantitative analysis of your fluorescent western blot can be performed immediately after imaging. Figure 2 shows the digital image of the western blot labeled with three different primary antibodies (anti-tubulin, anti-beta actin, and anti-GAPDH) and fluorescently labeled secondary antibodies. The individual channels are shown in grayscale and all three are merged in the three-color image (upper left). The ability to visualize three proteins at once increases the potential information that can be obtained from individual western blot experiments. With more information, better quantitative analysis can be performed – strengthening your data and your science. Figure 2. Digital image of 3-color western blot using Azure Biosytems c600 imager. Lanes (from left to right) loaded with 1, 2, 5, 10, 20 µg HeLa cell lysate. Probed for tubulin (top), beta actin (middle) and GAPDH (bottom). The following settings were used: Light sources 6/7/4; Exposure time 1s / 13 s 204 ms / 677 ms; Filter positions 6/7/4; Aperture 6400; Focus 5000 / 5250 / 5000; bin level 1x1. Primary Antibody Secondary Antibody Excitation/ Emission Azure Imaging Channel Rat anti-Tubulin AzureSpectra Goat-anti-rat 800 785 nm/ 815 nm IR800 Rabbit antibeta-actin AzureSpectra Goat-anti-rabbit 700 660 nm/ 720 nm IR700 Mouse antiGAPDH AzureSpectra Goat-antimouse 550 551 nm/ 565 nm Cy3 Product: cSeries | Application: Western blotting Phosphorylated protein detection is more efficient by fluorescent Western blot Introduction The detection of phosphorylated proteins usually requires showing both nonphosphorylated and phosphorylated species. For traditional chemiluminescent methods, the negligible size difference between the phosphorylated and nonphosphorylated protein of interest requires probing with a phospho-specific antibody, then stripping, and then re-probing with the protein-specific antibody. Along with potential negative effects on data quality, the process of stripping and re-probing can take a considerable amount of time and reagents. The drawbacks from the traditional chemiluminescent method of detection can be overcome by using fluorescent secondary antibodies. Fluorescent secondary antibodies bearing two different fluorophores (with nonoverlapping spectra) allow for multiplexing; this means both phosphorylated and non-phosphorylated species can be detected simultaneously.1 The ability to multiplex not only makes IR detection the faster way to visualize phosphorylated and nonphosphorylated versions of a protein of interest, but it also makes quantitation more accurate. Stripping a blot is not always even or completely effective; moreover, the process of stripping can sometimes take off some of the original protein.2 Quantitation of proteins before and after stripping is less accurate than if no stripping were involved. Thus, a protocol that visualizes modified and unmodified proteins at the same time offers improved accuracy as well as convenience of speed (see Table 1 for comparison). Methods Multiplexing protocol: Probing for two proteins of the same weight Untreated and IFNα-treated HeLa lysates were loaded on an SDS polyacrylamide gel and resolved by electrophoresis. The proteins were transferred to a PVDF membrane using Azure Transfer Buffer. The membrane was split and one membrane was probed using a chemiluminescent detection protocol while the other membrane was probed using a protocol with fluorescent antibodies as detailed below. Chemiluminescent detection After transfer, the first blot was probed for phosphoSTAT1. First the membrane was blocked for 30 minutes before incubation with 4 µg rabbit anti-phospho-STAT1 for 1 hour. The membrane was then rinsed twice with 25 mL Azure Fluorescent Wash Buffer (AFWB) followed by three 5 minute washes with AFWB. Next, it was incubated with 3 µg anti-rabbit-HRP for 1 hour, followed by a wash as before with AFWB. After washing, the blot was incubated with 10 mL of the chemiluminescent substrate Radiance for 2 minutes. The blot was imaged directly by the Azure cSeries. Time Investment for Western Blots Chemi Time (min) Fluorescent Time (min) Blocking 30 10 Primary incubation 60 60 15 15 60 60 15 15 Protocol steps Wash Secondary incubation Wash Add substrate 2 – PBS rinse or Water rinse – 5 5 – Stripping 5 Wash 15 Blocking again 30 Primary incubation 60 Wash Secondary incubation Wash 15 60 15 Add substrate 2 TOTAL TIME 6 hrs 29 min Table 1. Time required for each western blot protocol. 2 hr 45 min After imaging the blot, the probed membrane was stripped: first, the membrane was rinsed in high purity water for 5 minutes; second, the membrane was placed in Azure HRP Stripping Buffer for 5 minutes. Following stripping, the membrane was washed with 25 mL AFWB, 2 times fast and then 3 times for 5 minutes each. To probe for STAT1, the membrane was blocked again for 30 minutes and then incubated with 4 µg mouse antiSTAT1 for 1 hour. The membrane was then rinsed twice with 25 mL AFWB followed by three 5 minute washes with AFWB. Next it was incubated with 3 µg anti-rabbit-HRP for 1 hour, followed by a wash with AFWB in a similar manner as before. After washing, the blot was incubated with 10 mL Radiance for 2 minutes. The blot was imaged again by the Azure cSeries. Using fluorescent antibodies and Infrared (IR) detection After transfer, the membrane was blocked for 10 minutes at room temperature. To probe for the proteins of interest, the membrane was incubated with 4 µg rabbit antiphospho-STAT1 and 4 µg mouse anti-STAT1 for 1 hr. The membrane was then rinsed twice with 25 mL Azure IR Fluorescent Wash Buffer (AIWB) followed by three 5 minutes washes with AIWB. Following the wash, the membrane was incubated with 4 µg of anti-rabbit-800 and 4 µg anti-mouse-700 for 1 hour. The probed membrane was washed with AIWB in the same manner as before. After the AIWB wash, the blot was rinsed with 25 mL of PBS for 5 minutes before imaging. The blot was imaged with the Azure cSeries, using the filters IR-800 (green) and IR-700 (red). The comparisons presented here aim to demonstrate the improved convenience and accuracy of fluorescence detection as compared to traditional chemiluminescent methods. The main differences between methods include secondary antibodies used and the number of steps involved. The extra steps in chemiluminescent detection methods include stripping the membrane, which leaves the possibility for uneven removal of antibodies and possible loss of target proteins. Additionally, chemiluminescent detection required approximately 6.5 hours of time to attain data of both unmodified and phosphorylated STAT1. With fluorescent secondary antibodies, STAT1 and phospho-STAT1 could be imaged simultaneously with different IR filters in the digital imager in less than 3 hours (see Table 1). Both protocols can provide quality results, so it’s important to choose the protocol that best fits your needs.1,2 1 2 3 4 Phospho-STAT1 STAT1 Figure 1. Chemiluminescent western blot of STAT1 and phosphoSTAT1. The blot was first probed with anti-phospho-STAT1 and imaged. After stripping, the blot was re-probed with anti-STAT1 and imaged. Lanes: Ladder, 1) 10 µg untreated HeLa lysate, 2) 10 µg IFNα-treated, 3) 20 µg of untreated, 4) 20 µg IFNα-treated. Results and Conclusions In this note, two corresponding western blot membranes were subjected to either chemiluminescent or IR detection for analysis of phosphorylated STAT1. HeLa cells were either treated with IFNα to induce phosphorylation of STAT1 or left untreated, and then protein lysates were harvested and resolved by gel electrophoresis followed by transfer to a membrane for western blotting. Each blot was probed for STAT1 and phospho-STAT1 using the same primary antibodies, but secondary antibodies that were either HRP-conjugated (Figure 1) or fluorescently labeled (Figure 2) were used depending on the detection method. Figure 2. Fluorescent western blot of STAT1 and phospho-STAT1. The blot was probed with anti-phospho-STAT1 and anti-STAT1 followed by fluorescent secondary antibodies, and then imaged on Azure cSeries. Top right is the green channel, using IR-800; top left is the image of the red channel, using IR-700. Bottom image is both channels merged. Lanes are the same as in Figure 1. References Products and Reagents Product Number 1. Gingrich, J.C., Davis, D.R., Nguyen, Q. Multiplex Detection and Quantitation of Proteins on Western Blots Using Fluorescent Probes. BioTechniques. 2000. 29:636-642 Low Fluorescence PVDF Membrane AC2105 Azure Transfer Buffer AC2127 2. MacPhee, D.J. Methodological Considerations for Improving Western Blot Analysis. Journal of Pharmacological and Toxicological Methods. 2010. 61: 171-177. Azure Fluorescent Wash Buffer AC2113 Azure IR Fluorescent Wash Buffer AC2145 Goat-Anti Rabbit HRP Secondary Antibody AC2114 Goat-Anti Mouse HRP Secondary Antibody AC2115 AzureSpectra 700 Goat-anti-mouse Secondary Antibody AC2129 AzureSpectra 800 Goat-anti-rabbit Secondary Antibody AC2134 Radiance Chemiluminescent Substrate AC2101 Azure HRP Stripping Buffer AC2154 Product: cSeries | Application: Western blotting Imaging Three-color Western Blots with the Azure c600 Measuring two or more proteins is important for quantitative Western experiments. With multicolor detection, the amount of a protein of interest can be normalized to that of a housekeeping protein, allowing levels of the protein of interest to be compared between samples. Also, with multicolor fluorescent detection, proteins that are similar in size and not well separated spatially on a gel, such as phosphorylated and nonphosphorylated isoforms of a protein, can be measured at the same time. secondary antibody conjugate secondary antibody conjugate Dye 1 primary antibody (mouse) Dye 2 primary antibody (rabbit) membrane Figure 1 proteins transferred to the membrane blocking compound The Azure c600 imaging system is designed for multicolor fluorescent Western blotting, with two nearinfrared (NIR) and 3 visible fluorescent channels. This application note demonstrates a procedure to obtain three-color fluorescent blots using WesternDot® reagents. WesternDot® reagents are secondary antibodies conjugated to Qdot® nanocrystals enhanced with VIVID® technology. The WesternDot® reagents WesternDot® 585, WesternDot® 655, and WesternDot® 800 comprise an ideal combination for 3-color Western blotting experiments because these fluorophores have well-resolved, non- overlapping emission spectra (Figure 2). All WesternDot® reagents can be excited with blue or UV light (Figure 2), so imaging each protein independently on a multiplex fluorescent blot requires an instrument such as the Azure c600 that has emission filters specific for the emission of each WesternDot® reagent. Excitation (dashed line) Emission (solid line) Multicolor detection is a powerful application of fluorescent Western blotting. Two or more proteins can be detected on one blot using antibodies labeled with different fluorophores (Figure 1). The ability to simultaneously assay multiple proteins on a Western blot represents a significant advantage of fluorescent detection over chemiluminescent detection, because chemiluminescent detection requires stripping and re-probing a blot or using duplicate blots to detect two proteins, approaches that can introduce errors and variation. 350 WesternDot® 585 WesternDot® 655 WesternDot® 800 400 Figure 2 450 500 550 600 650 700 750 800 850 900 Wavelength (nm) Methods Approximately 10 μg each of cell lysates from cells induced to express EGFR or phosphorylated-AKT were mixed and loaded on a Novex® Bolt™ 4-12% Bis-Tris Plus 10-well gel. After electrophoretic separation, proteins were transferred to a nitrocellulose membrane using the iBlot® 2 Gel Transfer Device. Western blot processing was carried out with an iBind™ Western Device using the iBind™ FD Solution standard protocol. The membrane was probed with 2 mls of iBind™ FD solution containing the three primary antibodies: mouse anti-EGFR (diluted 1:200), rabbit anti–phospho-AKT (diluted 1:400), and chicken anti-GAPDH (diluted 1:400). Secondary antibodies used were WesternDot® 585 goat anti-mouse, WesternDot® 800 goat anti-rabbit, and WesternDot® 655 goat anti-chicken (Table 1). All secondary antibodies were diluted 1:100 in iBind™ FD Solution. The blot was imaged with four imaging systems, as shown in Table 2. Results The same three-color blot was imaged with four different imaging systems. The acquired images are shown in Figure 3. The Odyssey instrument (Figure 3a) does not have the capability to detect visible fluorescent dyes, so only one protein, phosphorylated AKT detected in the IR800 channel, is visible. The Typhoon instrument (Figure 3b) has settings for detection of Qdot® 800 and Qdot® 655, and a merged image of both channels is shown. Only GAPDH, detected at 655 nm, is visible; no signal from phosphorylated AKT is seen. The LAS-1000 instrument (Figure 3c) has a blue light source capable of exciting all three WesternDot® reagents, but only one emission filter so all fluorescent signals are detected in a single image. EGFR (detected at 585 nm) and GAPDH are visible, but phosphorylated AKT is not. Of the four imaging systems used, only the Azure c600 (Figure 3d) detected all three WesternDot® conjugates. The c600 has default settings for common fluorescent dyes and can also be manually configured. To image the WesternDot® conjugates custom settings were used; the blot was excited using the settings for imaging Cy2 (460 nm), and detected using three different emission filters (Table 2). All three channels are merged in the image shown (Figure 3d), with WesternDot® 655 shown in blue, WesternDot® 585 in red, and WesternDot® 800 in green. The ability to detect three proteins on a single blot dramatically increases the amount of information that can be obtained from a Western blot experiment. For example, two proteins and a loading control can be detected A B C Primary antibody Secondary antibody Excitation/Emission Mouse anti-EGFR WesternDot® 585 goat anti-mouse 405-485 nm/585 nm Rabbit anti-phosphoAKT WesternDot® 800 goat anti-rabbit 405-485 nm/800 nm Chicken anti-GAPDH WesternDot® 655 goat anti-chicken 405-485 nm/655 nm Table 1: Protein targets, primary antibodies, and secondary antibodies Imaging system EGFR GAPDH Phospho-AKT Odyssey (LI-COR) NA NA 800 channel Typhoon™ FLA 9000 (GE Healthcare) NA Qdot® 655, PMT 1000 Qdot® 800, PMT 1000 LAS-1000 (Fujifilm Life Science) Blue-light excitation 3-second exposure Blue-light excitation 3-second exposure Blue-light excitation 3-second exposure c600 (Azure) Excite Cy2/ detect orange filter Excite Cy2/ detect red filter Excite Cy2/ detect 800 filter (manufacturer) (WesternDot® 585) (WesternDot® 655) (WesternDot® 800) Table 2: Imaging systems and settings used for each target protein simultaneously, allowing quantitative comparisons to be made between proteins and between samples. WesternDot® reagents are available with a variety of species specificities, for flexibility in designing threecolor experiments. With the Azure c600 and WesternDot® reagents, three-color Western blots are at your fingertips. D EGFR 585 nm Phospho-AKT 800 nm GAPDH 655 nm Figure 3. Images of the same three-color Western blot, detected with WesternDot® reagents WesternDot® 585, WesternDot® 655, and WesternDot® 800, collected using four different imaging systems. a. LI-COR Odyssey; b. GE Healthcare Typhoon 9000; c. FUJIFILM LAS-1000; d. Azure c600. Product: cSeries | Application: Gel Imaging Superior electrophoresis results with Lonza reagents and the Azure cSeries imaging systems Introduction Lonza, a leader in electrophoresis reagents, and Azure Biosystems, manufacturer of gel doc and Western blot imaging systems, have teamed up to demonstrate how to easily obtain excellent DNA and protein gel electrophoresis data. Lonza provides a complete line of reagents for nucleotide and protein electrophoresis including precast gels, buffers, stains, and markers for fast and reliable electrophoresis. Using precast gels saves time and allows for reproducible results due to tight quality control. The cSeries family of digital imaging systems from Azure Biosystems offers a range of instruments to suit imaging needs from gel documentation to quantitative imaging of multicolor fluorescent Western blots. Each instrument is capable of imaging commonly used DNA and protein stains, having illumination sources that include both white light and dual-UV transilluminators, as well as epi white and blue light. The cSeries instruments include user-friendly interfaces to make capturing the best possible image a breeze. This application note demonstrates the high-sensitivity and quality results that can be obtained combining the expertise of these two companies for DNA and protein electrophoresis and analysis. offer multiple options for imaging the FlashGel™ dye; UV transillumination, UV transillumination with a blue conversion screen that essentially converts the light source to a blue transilluminator, and epi blue light. Figure 1 shows the excellent separation, sensitivity, and image quality obtained imaging a double tier FlashGel™ with the cSeries, using the UV transilluminator and blue conversion screen. The image was captured using a 10 second exposure. Fragments ranging from 50 bp to 1500 bp in size were well resolved on a 2.2% agarose gel in less than 5 minutes. High reproducibility is seen across the gel and across both tiers and the image demonstrates uniform intensity across the gel with excellent signal-to-noise. For samples loaded and imaging specifics, please see experimental details at the end of the application note. Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 bp 1500 150 50 bp 1500 150 50 Fast nucleotide electrophoresis and sensitive detection with the FlashGel™ System and cSeries imager Figure 1. FlashGel™ imaged with the Azure cSeries imager, using UV transilluminator with blue conversion screen. The FlashGel™ System from Lonza consists of precast agarose gel cassettes that are run in an accompanying dock. Electrophoresis does not require preparation of running buffer or a separate staining step. Each gel contains a proprietary stain that is 5 to 20 times more sensitive than ethidium bromide and that can be imaged using either blue light or UV light with the same settings and filters that would be used with ethidium bromide. The Azure Biosystems cSeries instruments Figure 2 compares the limit of detection achieved when imaging a FlashGel™ System with the cSeries imaging system using either UV transillumination with a blue light conversion screen (Figure 2A), UV transillumination (Figure 2B), or epi blue light (Figure 2C). Though imaging with the blue conversion screen provides the optimal balance of sensitivity and low background, in every image a band containing less than 0.2 ng of nucleic acid 0.156 ng 0.156 ng 0.156 ng 0.31 ng 0.31 ng 0.31 ng 1.25 ng 2.5 ng 0.625 ng 0.625 ng 0.625 ng 1.25 ng 1.25 ng 5 ng 5 ng 2.5 ng 2.5 ng 0.19 ng 0.325 ng 0.325 ng 1.5 ng 0.19 ng 5 ng 0.19 ng 0.325 ng 0.75 ng 1.5 ng 3 ng 3 ng C 1.5 ng 3 ng B 0.75 ng A 0.75 ng is easily detected. For details of samples loaded and imaging settings, please see experimental details at the end of the note. Accurate electrophoresis and sensitive detection of larger-format Latitude™ gels with the cSeries imaging system Latitude™ gels are precast agarose gels containing ethidium bromide. The gels are precision cast for high accuracy and reproducibility. Figure 3 shows a midi 1% SeaKem™ LE Plus gel imaged using a cSeries imager and UV transilluminator. The large imaging area of the cSeries instrument is well able to handle the largerformat gel (10x15 cm) and the even lighting and exposure reveal the highly reproducible electrophoresis of samples both across the entire gel and between the top and bottom set of wells. For details of samples loaded and imaging settings, please see experimental details at the end of the note. Figure 3. Imaging a Latitude™ Midigel with the Azure cSeries. cSeries imaging of protein gels stained with ProSieve™ EX Safe Stain Figure 2. Comparison of sensitivity obtained imaging a FlashGel™ with the Azure cSeries imager using either (A) UV transilluminator with blue conversion screen, (B) UV transilluminator (UV 302), or (C) epi blue light. For routine protein electrophoresis, PAGEr™ Gold precast Tris-glycine gels provide sharp resolution in a convenient, easy-to-use package. Figure 4 shows a PAGEr™ Gold gel stained with Lonza’s ProSieve™ EX Safe Stain and imaged using white light transillumination on the Azure cSeries imager. The image demonstrates high sensitivity and excellent signal-to-noise. For details of samples loaded and imaging settings, please see experimental details at the end of the note. Figure 5 demonstrates the limit of detection for a PAGEr™ Gold gel stained with ProSieve™ EX Safe Stain. A series of two-fold serial dilutions of carbonic anhydrase were loaded on the gel. A band containing less than 10 ng of protein is easily visualized in the image. 1 2 3 4 5 6 7 8 9 10 11 12 Figure 4. PAGEr™ Gold protein gel stained with ProSieve™ EX Safe Stain and imaged using the white light transilluminator of the Azure cSeries. 1 2 3 4 5 6 7 8 9 10 11 Figure 5. High sensitivity of ProSieve™ EX Safe Stain-stained gel imaged with the Azure cSeries. Maximize convenience without sacrificing quality The data presented demonstrate the high-quality results that result from combining the convenience of Lonza’s precast gels and other reagents with the flexible, highresolution imaging of the Azure cSeries imaging systems. The cSeries imagers provide light sources and filters compatible with all commonly used nucleotide and protein stains, including the proprietary stain included in Lonza’s nucleotide FlashGel™ as well as Lonza’s ProSieve™ EX Safe Stain for protein. The large imaging area of the cSeries instruments allows imaging of both small and large gels and the user-friendly interface makes image capture a breeze. To learn more about Lonza electrophoresis products please visit the Lonza website and to learn more about the cSeries imaging systems please visit www.azurebiosystems.com. Experimental details Figure 1 DNA samples were separated on a 2.2% 16+1 well double tier FlashGel™ DNA Cassette (Lonza product #57032). The samples loaded were as follows: lane 1, 4 µL 50-1500 bp FlashGel™ Marker (Lonza product #57033); lane 2, 4 µL FlashGel™ QuantLadder (Lonza product #50475); lane 3, 5 µL 150 bp DNA Fragment; lane 4, 5 µL 250 bp DNA fragment; lane 5, 5 µL 350 bp DNA fragment. The samples were then repeated across the gel. DNA fragments were diluted in 1X FlashGel™ Sample Buffer (Lonza). The gel was run for 4.5 minutes at 275 V. Imaging was conducted with a cSeries imager using the UV transilluminator at 302 nm with blue light conversion screen (Azure SKU AC1025). Exposure was 10 seconds. Figure 2 DNA samples were separated on a 2.2% 12+1 well single tier FlashGel™ DNA Cassette (Lonza product #57031). The samples loaded were as follows: lane 1, 50-1500 bp DNA markers (Lonza product #57032); lanes 2-6, serial two-fold dilutions of FlashGel™ QuantLadder (Lonza product #50475) starting with 3 ng of the smallest (100 bp) band in lane 2; lanes 7-12, serial two-fold dilutions of a 400 bp DNA fragment starting with 5 ng in lane 7; lane 13, 50-1500 bp DNA marker. The gel was run for 8 minutes at 275 V. Imaging with UV transillumination and blue conversion screen (Azure SKU AC1025) was conducted using a 10 second exposure. Imaging with UV transillumination (302 nm) was conducted using the AutoExposure function of the cSeries. Imaging with epi-blue light was conducted with an exposure time of 2 seconds. Figure 3 DNA samples were separated on a 1% SeaKem LE Plus gel containing ethidium bromide (Lonza product #57230). Samples loaded were as follows: lane 1, 4 µL 1-10 kb DNA Marker (Lonza product #50471); lane 2, 8 µL FlashGel™ 100-4000 bp DNA Marker (Lonza product #50473); lane 3, 8 µL FlashGel™ QuantLadder (Lonza product #50475); lane 4, SimplyLoad™ 100 bp DNA ladder (Lonza product #50327); lane 5, SimplyLoad™ 500 bp DNA ladder (Lonza product #50329); lane 6, 1 kb DNA fragment (BioVentures); lane 7, 2 kb DNA fragment (BioVentures); lane 8, 4 kb DNA fragment (BioVentures). The samples were then repeated across the gel. The gel was set up in an Owl B2 chamber with 1X TBE (with 0.5 µg/mL EtBr). The gel was run at 115 V (approximately 5 V/cm) for 85 minutes. The gel was imaged using UV transillumination at 302 nm. Exposure time was 6 seconds. Figure 4 Figure 5 Protein samples were separated on a 4-20% PAGEr Gold gel (Lonza product #58105). Samples loaded were as follows: lane 1, 5 µL PageRuler Marker (Thermo Fisher); lane 2, 4 µL ProSieve™ Unstained Marker (Lonza); lane 3, 1 µg ß-galactosidase; lane 4, 2.5 µg Phosphorylase B; lane 5, 2 µg BSA; lane 6, 2 µg ovalbumin; lane 7, 2 µg carbonic anhydrase; lane 8, 4.5 µL E. coli LE 392 lysate; lane 9, 5 µL ProSieve™ QuadColor™ Protein Marker (Lonza product #193837); lane 10, 6.5 µL ProSieve™ Color Marker (Lonza); lane 11, 5 µL PageRuler Marker (Thermo Fisher); lane 12, 3 µL ProSieve™ Unstained Marker (Lonza). The gel was run for 40 minutes at 200 V. After electrophoresis, gel was placed immediately into ProSieve™ EX Safe Stain (Lonza product #00201455), heated in a microwave for 55 seconds, then stained with shaking for 20 minutes. The gel was then destained by pouring off the stain, adding water, and warming in a microwave with a piece of paper towel to absorb excess stain. The gel was imaged on the cSeries imager using the White Light Table (Azure SKU AC1029) and default settings for aperture and focal height. ™ Protein samples were separated on a 4-20% PAGEr™ Gold gel (Lonza product #58105). Samples loaded were as follows: lane 1, PageRuler Marker (Thermo Fisher); lanes 2-10, two-fold serial dilutions of carbonic anhydrase starting with 2 µg of protein in lane 2; lane 11, ProSieve™ QuadColor™ Protein Marker (Lonza product #193837). The gel was run for 40 minutes at 200 V. After electrophoresis, the gel was stained, destained, and imaged as described for Figure 4. Product: cSeries | Application: Gel imaging DNA Dye Detection Limits using Azure cSeries Imagers Introduction Gel Casting and Running Gel-based detection of DNA was first described in 1972 by Aaij and Borst1 and has gone on to become a cornerstone technique of molecular-biology. With the development of increasingly advanced and sensitive DNA technologies, reliable, accurate quantification of low concentration DNA products is becoming increasingly desirable. 0.8% agarose gels in 1x TAE were cast, 10 µL of each dilution was loaded and gels were run in 1x TAE for 120 to 150 minutes at 70V. One gel was cast with EtBr at a dilution of 1:10,000, all other gels were cast without dye and post-stained. Post-staining was performed with a 1:10,000 dilution of either EtBr, EZ-Vision® Bluelight, SYBR® Gold, SYBR® Green or SYBR® Safe in TAE buffer for 30 minutes. The traditional method for gel-based DNA detection is to add ethidium bromide (EtBr) during gel casting. However, the use of EtBr is falling out of favor due to the mutagenic characteristics of EtBr arising from its intercalation of double stranded DNA which may affect DNA replication or transcription. Additionally, although EtBr is not classified as hazardous waste at low concentrations many organizations treat it as such, often at great expense. Whilst staining of gels post-run with EtBr can reduce the amount of hazardous waste generated, it does not address issues arising from exposure of workers to a mutagen. Because of this a variety of non-mutagenic DNA dyes have been developed; however, unless they are capable of matching, or improving on the sensitivity of EtBr then their use is unlikely to gain traction. With the rise and advancement of digital imaging technology and techniques, the use of non-mutagenic DNA dyes (“safe” stains) has increased in popularity do to their combined ease of use and dynamic range. Therefore, we assessed the limit of detection (LoD) for various DNA dyes using cSeries imagers from Azure Biosystems. Methods Sample Preparation New England Biolabs Quick Load 1kb DNA Ladder, which features known concentrations of DNA for each band, was serially diluted 1:1 in water and 6X gel loading dye (See Table 1 for dilutions). Gel Imaging Following staining, gels were imaged on both the Azure c200 and c600 imagers. Gels stained with EtBr were imaged using the UV transilluminator (302 nm), all other gels were imaged using Epi-Blue lights with an orange filter. In all instances images were captured using autoexposure settings. No difference in sensitivity was detected between images generated by the Azure c200 or c600 confirming that any limit of detection differences are dye dependent. Band 4kb Lane 3kb 2kb Mass (pg) in 10 µL 1 4125 15625 6000 2 2063 7813 3000 3 1031 3906 1500 4 516 1953 750 5 258 977 375 6 129 488 188 7 64 244 94 8 32 122 47 Table 1. DNA ladder dilutions. Results and Conclusion Band In this note the sensitivity of various DNA dyes was assessed. A 1kb DNA ladder of known concentration was serially diluted and samples were resolved by agarose gel electrophoresis. One gel was cast with EtBr with all others post-stained. Sample gel images displaying the 2kb band are shown (Figure 1) along with a summary of DNA dye LoDs (Table 2). Stain Briefly, post-staining of gels with EtBr did not provide the same LoD as casting gels with EtBr in situ. All alternative DNA dyes displayed a similar LoD to EtBr post-stained when observing larger DNA fragments. Whilst EtBr displayed an enhanced LoD, when detecting smaller fragments, compared to EZ-Vision® Bluelight, SYBR® Gold and SYBR® Green; SYBR® Safe demonstrated an identical LoD. 3kb 2kb Limit of Detection (pg) EtBr Cast in Gel 32 122 47 EtBr post-stained 64 244 94 EZ-Vision® Bluelight 32 244 188 ® SYBR Gold 32 122 94 SYBR Green 32 244 94 SYBR Safe 32 122 47 ® ® Table 2. Summary of DNA dye LoDs. Gray shading indicates highest limit of detection. Lane Mass (pg) 1 2 3 6000 3000 1500 4 750 5 375 6 188 7 94 8 47 EtBr Cast in Gel These results suggest that labs looking to reduce worker exposure to mutagenic EtBr whilst also reducing the amounts of hazardous waste produced do not have to accept a DNA dye with a lower limit of detection. EtBr Post-stained Reference SYBR® Gold 1. Aaij, C. & Borst, P. The gel electrophoresis of DNA. Biochim Biophys Acta 269, 192-200 (1972). 4kb EZ-Vision® Bluelight SYBR® Green SYBR® Safe Figure 1. Samples gel images. Representative blots showing 2kb band of DNA ladder. Product: cSeries | Application: Fluorescent imaging Imaging Viral Load in Chicken Embryos Introduction Results and Conclusions Newcastle Disease Virus (NDV) is a contagious disease, capable of transmission to humans, and with a high risk of a severe epizootic in birds, particularly domestic poultry which are typically reared at high density. Figure 1 shows an embryo infected with a nonvirulent form of the virus next to an uninfected embryo (negative control). The infected embryo shows the non-virulent strain of the virus confined primarily in the umbilical tissue. While the effects of NDV are mild in humans, its effect in avian hosts are more pronounced and vary based on the age and species of the host, as well as the strain of the virus. Three strains of NDV, with differing levels of virulence, have been well characterized in chicken eggs. Strains of intermediary virulence are known to infect primarily the umbilical tissue of the chicken embryo, whilst more virulent strains infect other tissues indiscriminately. As such many studies have been performed to investigate the impacts of the varying strains along with the mechanisms of viral load in domestic chickens. However, such studies have often been limited in their ability to accurately and rapidly assess viral load in vivo. With the development of sensitive high-resolution imaging systems, such as the Azure c600, it is now possible to quickly and easily image fluorescently tagged NDV in chicken embryos, and, to some extent, quantify the viral load in various embryonic tissues. Methods Figure 1. NDV eGFP No-VIR (left) and Negative Control (right). Figure 2 shows the same non-virulent virus-infected embryo imaged alongside an embryo infected with a more virulent strain of NDV. The virulent form of the virus infects the embryo more indiscriminately, and the virus is detected throughout the embryo. Chicken embryos were infected at E10 and incubated for a further 72 hours. Embryos were infected with either 100-1000 PFU of virulent or non-virulent recombinant NDV tagged with eGFP, and compared with a noninfected control. The embryos were placed on petri dishes and imaged with the Azure c600 using Epi Blue imaging mode with an automated capture. Figure 2. NDV eGFP No-VIR (left) and NDV eGFP VIR (right). Figure 3 shows the extent of the virulent infection compared to the negative control, uninfected embryo. Conclusions The utility of fluorescent imagers like the Azure Biosystems c600 extends far beyond Western blotting, including a large number of applications that utilize fluorescence excitation. This application note showcases one such application – imaging a fluorescently tagged virus in animal models allowing for the generation of high-resolution quantifiable images. Acknowledgements Thank you to Ray Izquierdo-Lara, Ana Chumbe, Katherine Calderon, Manolo Fernandez. FARVET SAC, Peru, for providing the images. Figure 3. NDV eGFP VIR (left) and Negative Control (right). Product: Sapphire | Application: Phosphor imaging Phosphor Imaging with the Sapphire Biomolecular Imager Introduction Results and Conclusions While fluorescent and chemiluminescent imaging techniques and their detectors are becoming more sensitive and selective, photostimulated luminescence from phosphor storage plates remains a key application for many researchers, especially for applications such as electrophoretic mobility shift assays (EMSA), enzyme assays, and in vivo imaging. The Sapphire Biomolecular Imager captures high quality images with a large dynamic range from standard storage phosphor screens (Figure 1). Following sample quantification using AzureSpot analysis software, the LOD for American Radiolabeled Chemicals 14C standard after a 3-hour exposure was determined to be 0.036 μCi/g. Furthermore, a highly linear dynamic range from 1000 to 0.036 μCi/g was observed (r2 = 0.99 [Figure 2]). Traditional phosphor imagers are often single function and require a large footprint. The Azure Biosystems Sapphire Biomolecular Imager offers the ability to scan storage phosphor screens for filmless autoradiography, with exceptional dynamic range and image quality thanks to the use of laser excitation and photon multiplier tube (PMT) detection. The compact Sapphire Biomolecular Imager also incorporates exceptional visible fluorescent and near infra-red scanning along with true chemiluminescent and white light imaging making it a true lab workhorse. To conclude, the Sapphire Biomolecular Imager is capable of imaging storage phosphor screens producing high quality images for later quantification; while also offering the ability to rapidly image fluorescent, chemiluminescent and NIR samples all within a single compact footprint. 0.036 4.5 µCi/g Materials and Methods An American Radiolabeled Chemicals 14C standard with slices from 1000 to 0.004 μCi/g was exposed to a BAS-MS storage phosphor screen (0.9 DPM/mm2/hr sensitivity) for 3 hours. Storage Phosphor Plate Imaging Following exposure, the storage phosphor screen was imaged using the Phosphor Imaging module of the Sapphire Biomolecular Imager with standard excitation and detection settings. Samples were quantified and limits of detection (LOD) and dynamic range (DR) calculated. Figure 1. American Radiolabeled Chemicals Carbon-14 standard exposed to Storage Phosphor Screen for three hours then imaged on the Sapphire Biomolecular Imager. 5 Log Integrated Intensity Sample Preparation 4 3 2 1 0 1 2 3 4 5 6 Log nCi/g Figure 2. Sample: 14C autoradiographic standard; Imaging: Phosphor; LOD: 0.036 µCi/g; DR: 5.4 orders of magnitude; Linearity: R2=0.99 Step Product Part number Sample preparation American Radiolabeled Chemicals – 14C standard ARC0146F BAS-IP-MS – Storage phosphor screen IS1011 Cassette for Phosphor Imaging IS1012 Azure Biosystems – Sapphire Biomolecular Imager with Phosphor Imaging Upgrade IS1002, IS1004 Storage phosphor plate Phosphor plate imaging Table 1. Materials and product numbers. Product: Sapphire | Application: 96 well plate imaging, In-cell imaging Detecting Proteins In-Situ with In-Cell Western Blotting Introduction Western blotting was first described by three groups in 1979. Since then numerous refinements to the basic technique, reagents used and imaging technologies have massively broadened the usage of Western blotting, making it a cornerstone protocol in life sciences today. However, the general workflow of a Western blot has remained the same throughout this time. Samples are isolated and protein is extracted and standardized. Proteins are then separated by gel electrophoresis, transferred and probed with primary and secondary antibodies before being visualized. With numerous steps, Western blotting as a protocol requires a large investment in equipment, training, reagents and, perhaps most importantly, time. A standard Western blot protocol takes two days to work from sample to image. Therefore, when analyzing a large number of samples or performing optimizations, it is easy to see how Western Blotting can quickly become a bottleneck in your research workflow. Enter in-cell Western blotting – a radical rethinking of the standard protocol coupling the ability to accurately quantify intracellular proteins from Western blotting with the repeatability, quick turnaround and highthroughput of an ELISA. Cells are grown on sterile plates, before being fixed and permeabilized in situ. Subsequent labelling is performed as normal with a specific primary antibody followed by an isotype specific fluorescent conjugated secondary antibody. Conjugates in the nearinfrared (NIR) spectrum are commonly used to reduce auto-fluorescence and noise typically associated with tissue culture plastics. The Azure Biosystems Sapphire™ Biomolecular Scanner is specifically designed to allow for visualization in two NIR channels (658 nm and 785 nm) as well as visible fluorescent wavelengths (488 nm and 520 nm), allowing for multiplex imaging within wells. This multiplex capacity allows for the assessment of multiple proteins of interest in a single well which is useful for looking at total and phosphorylated versions of a protein or to detect an in-well standard to allow for quantification across a plate. With a laser based excitation system using photomultiplier tube (PMT) and avalanche photodiode (APD) detectors, signal selectivity, sensitivity and speed of imaging achievable by the Sapphire make it an ideal choice for in-cell Western blotting. Materials and Methods Culture cells HeLa cells were serially diluted and seeded into a sterile 96 well tissue culture plate at a volume of 0.2 mL per well, and grown until approximately 80% confluent. All wells were fixed and permeabilized using 100% methanol for 15 minutes at room temperature. In-cell Western blotting Figure 1. Representative image of an in-cell Western blot. Following fixation and permeabilization, cells were rinsed in PBS, blocked with 1% fish gelatin in PBS for one hour at room temperature, then probed for alphatubulin and beta-actin overnight at 4°C. Samples were washed three times with PBS prior to incubation with AzureSpectra 550 and AzureSpectra 800 conjugated secondary antibodies for 60 minutes. Secondary antibodies were incubated along with RedDot™1 Nuclear Stain to normalize for total cell number. Plates were washed as previously described before imaging. Imaging After washing the plate was imaged on the Azure Biosystems Sapphire™ Biomolecular Scanner using the Plate focus setting. Results and Conclusions In this note we demonstrate the ability to quantify intracellular proteins in situ using the Azure Biosystems Sapphire™ Biomolecular Scanner. HeLa cells were grown in a 96-well plate probed with alpha-tubulin and beta-actin antibodies followed by isotype appropriate secondary antibodies conjugated with AzureSpectra 550 and AzureSpectra 800 respectively. All wells were incubated with RedDot™1 Nuclear Stain to normalize for total cell number. Images were acquired using an Azure Biosystems Sapphire™ Biomolecular Scanner and are displayed in Figure 2. The representative image shown demonstrates the high level of sensitivity and specificity achievable. Performing in cell Western blotting on 96 well plates allows for accurate measurement of intracellular proteins expression in situ; and provides a high throughput methodology to assess multiple stimulations, end-points, proteins of interest and replicates on a single plate. By using NIR antibodies and the Azure Biosystems Sapphire™ Biomolecular Scanner the potential for in well multiplex analysis also exists offering further improvements to throughput. 1 2 3 4 5 6 7 Figure 2. A serial dilution of HeLa cells were seeded into a 96-well plate, cultured, fixed and permeabilized. A) Columns 1-3 were probed for beta-Actin using AzureSpectra 550 (green). B) Columns 3-6 were probed for Tubulin using AzureSpectra 800 (blue). C) The entire plate was stained with RedDot1 Nuclear Stain as a normalization control (red). D) The individual channels were scanned simultaneously then combined into a single composite image using the Sapphire Capture Software. Product: Sapphire | Application: Western blotting Increasing Assay Efficiency with Four-Color Detection Introduction 4-color Western blotting The field of Western blot multiplexing – the ability to probe for multiple proteins on a single blot simultaneously – is developing rapidly. Chemiluminescent assays allowed for the detection of a single protein followed by multiple rounds of time consuming stripping and re-probing, with associated potential loss of protein and corresponding signal. Early Western blot multiplex imaging systems allowed for the imaging of two spectrally distinct fluorophores on a single blot. Researchers rapidly used this technique to assay for loading controls and proteins of interest on a single blot, to compare two distinct proteins, and to devise many other methodologies which are discussed in other application notes. Following transfer, the membrane was blocked for 30 minutes with Azure Fluorescent Blot Blocking Buffer then probed with rat anti-tubulin (Green), rabbit antibeta-actin (Red), and chicken anti-GAPDH (Gray) primary antibodies; and anti-transferrin (Blue) which had previously been labelled with AzureSpectra 490 dye using the AzureSpectra Labeling Kit. We have previously described an improvement on this two-channel methodology by imaging three proteins simultaneously by combining the fluorescent and nearinfrared (NIR) imaging capabilities offered by the Azure Biosystems c600 digital imager. In this note we discuss a further improvement – four-color Western blot multiplexing using the Azure Biosystems Sapphire™ Biomolecular Imager. The ability to simultaneously image four colors at one time greatly increases Western blot efficiency and the ability to make meaningful quantitative comparisons. Four-color Western blotting is made possible through the use of four spectrally distinct fluorophores and the Sapphire’s selective laser based excitation and sensitive photomultiplier tube (PMT) and avalanche photodiode (APD) detection systems. Materials and Methods Run and transfer gel Samples of 1.25 to 5 µg of HeLa cell lysate, some spiked with varying amounts of transferrin were electrophoresed on a 4-15% Tris-Glycine gel. After electrophoresis and separation, proteins were transferred to a low fluorescence PVDF membrane using Azure Transfer Buffer. Blots were rinsed and washed with Azure Fluorescent Blot Washing Buffer before being incubated with AzureSpectra labeled secondary antibodies – goat anti-rat 550 (Green), goat anti-rabbit 700 (Red), and goat anti-chicken 800 (Gray). After incubation, the blot was washed as before in Azure Florescent Blot Washing Buffer followed by a final rinse in PBS. Image 4-color Western blot After rinsing in PBS, the blot was allowed to dry before imaging on the Azure Biosystems Sapphire™ Biomolecular Imager. Results and Conclusions In this note a single Western blot was probed for four proteins simultaneously. HeLa cell samples, or HeLa cell samples spiked with transferrin were probed with tubulin (Green), beta-actin (Red) and GAPDH (Gray) antibodies followed by isotype appropriate secondary antibodies; as well as transferrin (Blue) antibody previously conjugated with AzureSpectra 490 dye for direct analysis. The Western blot was imaged using the Azure Biosystems Sapphire™ Biomolecular Scanner and Figure 1 shows the grayscale image captured at each wavelength for transferrin (a), tubulin (b), beta-actin (c) and GAPDH (d); together with a merged, colorized image (e). Together the images demonstrate the high level of sensitivity, specificity and lack of background signal it is possible to achieve using this methodology which allows for rapid and accurate quantitative analysis. The ability to image four proteins on a single blot greatly increases the efficiency of Western blotting, saving time and precious samples and allows for better quantitative analysis. The methodology also allows for the use of novel assays such as investigations of protein:protein interactions or on-blot total vs phosphorylated protein assays. a 490 – transferrin (blue) b Step Electrophoresis & transfer Blocking & antibody labelling 700 – actin (red) d Part number 4-15% Tris-Glycine gel N/A PVDF Membrane Azure, AC2105 Azure Transfer Buffer Azure, AC2127 AzureSpectra Fluorescent Blot Blocking Buffer Azure, AC2190 AzureSpectra Labeling Kit – 490 Azure, AC2185 Primary Antibodies Per protein of interest AzureSpectra Goat-anti-rat-550 Azure, AC2162 AzureSpectra Goat-anti-rabbit-700 Azure, AC2128 AzureSpectra Goat-anti-chicken-800 Azure, AC2137 PBS N/A 550 – tubulin (green) Probe Blot c Product 800 – GAPDH (gray) Table 1. Material and product numbers. e Figure 1. Digital images of 4-color Western blot, captured using Azure Biosystems Sapphire™ Biomolecular Imager. Transferrin (a), tubulin (b), actin (c) and GAPHD (d) were probed on a single blot using distinct fluorescent and near-infrared targeting antibodies. Images were captured using the Azure Biosystems Sapphire™ Biomolecular Imager at the specified wavelengths and merged into the four-color multiplex image (e). Sensitive and specific detection of all four proteins was possible with no evidence of background auto-fluorescence or bleed between channels. Product: AzureRed | Application: Western blotting Accurate Western Blot Normalization with AzureRed Fluorescent Protein Stain Introduction An important component of quantitative Western blotting is normalization of the band intensity obtained from the target protein against a reference whose intensity should only vary proportionally to the quantity of material in the sample. Traditionally, “housekeeping proteins” – proteins thought to be consistently expressed across different sample types – are used as such a reference. However, in some cases, housekeeping proteins may not be expressed consistently between samples. Studies have emerged showing that experimental conditions may alter the expression of some commonly used housekeeping proteins.1 In other situations, housekeeping proteins may not be present in the sample at a level that is similar enough to the protein of interest to fall within the linear range of detection.2-3 An increasingly popular alternative normalization method that avoids many of the drawbacks of relying on housekeeping proteins is “total protein normalization” or TPN. In this method, the volume of the protein of interest is compared to the total amount of protein loaded in the lane. TPN presents advantages over using a single protein as a standard including not being susceptible to unexpected changes in the amount of the standard protein between samples. Instead of looking at the intensity of one protein, the total density for each lane is measured. Because TPN technology eliminates many of the issues related to signal normalization, publications are increasingly requiring the use of total protein signal for normalization.4 AzureRed Fluorescent Protein Stain is a quantitative total-protein stain for gels and blots that is compatible with downstream protein analysis, including Western blotting. AzureRed is a perfect choice for staining applications, including post-transfer staining to confirm uniform protein transfer from gel to membrane, and staining quantitative Western blots as part of a TPN protocol. In this application note, we demonstrate that AzureRed Fluorescent Protein Stain is ideally suited for TPN because of its wide, linear dynamic range and sensitivity. Additionally, it is fully compatible with standard Western blot workflows, which we demonstrate by normalizing a tricolor fluorescent Western blot (three different proteins detected) to total protein using AzureRed stain. HeLa cell lysate, µg 2 4 6 8 10 12 Tubulin ß-actin GAPDH Figure 1. AzureRed is imaged simultaneously with three proteins of interest. The gel was loaded with dilutions of HeLa cell lysate. After transfer, the blot was stained with AzureRed and then probed for tubulin, ß-actin, and GAPDH without a destaining step. The blot was scanned with each of the four lasers of the Sapphire Biomolecular Imager. In this overlay of the four channels, total protein (AzureRed stain) is shown in gray; tubulin in red, ß-actin in blue, and GAPDH in green. Methods Samples of HeLa cell lysate were separated by polyacrylamide gel electrophoresis and proteins transferred from the gel to a low-fluorescence PVDF membrane (Azure Biosystems product AC2127; AC2105). After protein transfer, the blot was stained with AzureRed Fluorescent Protein Stain (Azure Biosystems product AC2124), according to the PVDF staining protocol (available at www.azurebiosystems.com). Following transfer, the blot was washed briefly in water then stained in AzureRed stain solution according to the protocol. After the 30-minute staining procedure, the blot was blocked using Azure Fluorescent Blot Blocking Buffer (Azure Biosystems product AC2190) and incubated with primary antibodies for Western blot detection (Table 1). HeLa cell lysate, µg A 2 4 6 8 10 B 12 2 4 6 8 10 12 Tubulin C The blot was imaged using the Sapphire Biomolecular Imager by scanning the blot with all four laser simultaneously. The three target proteins were detected with the lasers as shown in Table 1, while AzureRed staining was detected using the 520nm laser. B-Actin D GAPDH Target Primary antibody Secondary antibody Detected using laser E Tubulin Rat anti-tubulin Goat anti-rat AzureSpectra 700 658nm ß-actin Rabbit anti–ß-actin Goat anti-rabbit AzureSpectra 490 488nm GAPDH Chicken anti-GAPDH Goat anti-chicken AzureSpectra 800 Integrated intensity Images were analyzed using AzureSpot software. 784nm • • • • AzureRed Actin Tubulin GAPDH Figure 1. Overview of a Western blotting protocol using indirect antigen detection. Scanning the tricolor Western blot stained with AzureRed Fluorescent Protein Stain on the Sapphire Biomolecular Imager results in a four-channel composite image (Figure 1). All four channels are imaged and aligned as part of image capture, removing the need to align the images at a later point, and not adding any additional imaging steps in the workflow. The composite image with an overlay of the four resulting images allows easy comparison of the three proteins with each other and with total protein load. Viewing the single channel images allows visual inspection of the dynamic range of AzureRed and the three proteins (Figure 1A-D). The signal intensity for total protein in each lane (for AzureRed stain) or band (for each of the three proteins detected) was quantified using AzureSpot software. Figure 2E shows the relationship between each signal and the amount of protein loaded on the gel. The quantitative nature of AzureRed stain is observed in the excellent correlation of signal to amount of protein loaded (R2 = 0.994). This correlation was superior to that of any of the three individual proteins (Figure 2E), each of which is frequently used as a housekeeping protein. Figure 2. AzureRed has a wider linear dynamic range compared to common housekeeping proteins. Single color channel images for A) AureRed staining for total protein B) Tubulin C) B-actin and D) GAPDH. The superior dynamic range of the AzureRed signal to the actual amount of protein loaded is displayed in image (A). The signals from the total protein and the housekeeping proteins were quantified using the AzureSpot software, and the resulting values are plotted against the amount of protein loaded for each sample (E). AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins. n Integrated intensity Results Protein load (µg) n Raw Volume Normalized Volume Lane Figure 3. Tubulin signal normalized to total protein as assessed using AzureRed Fluorescent Protein Stain. The tubulin band was quantified using AzureSpot software. Raw tubulin band volume (blue bars) and band volume normalized to total protein (orange bars) are shown. Normalizing the signal volume using AzureRed corrects for any errors in sample loading or differences in transfer across the membrane. The use of the AzureRed signal for total protein as a standard to normalize protein signal is shown in Figure 3. Normalizing the tubulin signal for each lane, by the total protein signal for the same lane, results in excellent agreement across samples, with a coefficient of variation of normalized values of only 4%. Traditional Western Workflow Western Workflow with AzureRed TPN Separate proteins on gel Separate proteins on gel Transfer to membrane Transfer to membrane Stain for total protein with AzureRed Conclusion AzureRed Fluorescent Protein Stain is an ideal choice for quantitative fluorescent Western blotting. The wide, linear dynamic range ensures that AzureRed acts as an effective standard to account for variations in protein load. A key feature of AzureRed is the simplicity of fitting it into your existing workflow (Figure 4). AzureRed dye can be detected simultaneously alongside commonly used fluorescent probes without interference, allowing multiple proteins to be assessed on a single blot. Using the quantitative AzureRed signal for total protein normalization of multicolor blots makes it possible to quantify multiple proteins on a single blot without stripping or destaining. The protocol is quick, and imaging occurs simultaneously with the proteins of interest. Learn more about AzureRed Fluorescent Protein Stain at www.azurebiosystems.com. References 1. R.  E. Ferguson, H. P. Carroll, A. Harris, E. R. Maher, P. J. Selby, R. E. Banks, Proteomics 2005, 5, 566. 2. J.  Petrak, R. Ivanek, O. Toman, R. Cmejla, J. Cmejlova, D. Vyoral, J. Zivny, C. dD. Vulpe, Proteomics 2008, 8, 1744. 3. M.  Wilhelm, J. Schlegl, H. Hahne, A. M. Gholami, M. Lieberenz, M. M. Savitski, E. Ziegler, L. Butzmann, S. Gessulat, H. Marx, T. Mathieson, S. Lemeer, K. Schnatbaum, U. Reimer, H. Wenschuh, M. Mollenhauer, J. Slotta-Huspenina, J.-H. Boese, M. Bantscheff, A. Gerstmair, F. Faerber, B. Kuster, Nature 2014, 509, 582. 4. A.J.  Fosang, R.J. Colbran, Journal of Biological Chemistry, 2015, 290, 50. Block membrane Block membrane Western blot protocol Western blot protocol Image Image Figure 4. Total protein normalization with AzureRed fits easily into any Western blotting workflow.