Azure BiosystemsFluorescent Western blotting uses secondary antibodies directly conjugated to fluorescent dyes. Fluorophores for fluorescent Western blotting can be chosen based on their specific excitation and emission spectra, enabling multiplexing (detection of multiple proteins simultaneously, Figure 1) for faster, more efficient studies. One of the biggest advantages of fluorescent detection versus chemiluminescence is the ability to use more than one antibody per assay (see the Table).
With multiplexing, normalization and loading controls can be imaged at the same time and on the same blot as the sample. In addition, the ability to use different fluorophores enables visualization of proteins that are not well-separated electrophoretically, for more convenient imaging of the same protein with and without post-translational modifications.
Why would you need to visualize multiple proteins at once (Table 1). 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. Quantitation is also 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, multiplex Western blotting is possible and easy with AzureSpectra labeled antibodies and the Azure 600 Imaging System.
| Advantages | Disadvantages |
|---|---|
| Multiplex capability: multiple samples can be analyzed under the same experimental conditions. | Can be less sensitive than chemiluminescence |
| Increased quantitative accuracy | Membranes' auto fluorescence can increase background |
| Fluorescent label stability allows blots to be stored and re-imaged later | |
| More readout consistency using fluorescent dyes vs. the variability of an enzymatic reaction | |
| Cost and time savings |
Table 1. Advantages and disadvantages of multiplex fluorescent Western blots
Fluorescent Western blots are the gold standard for quantitative Westerns. Chemiluminescent Westerns are limited by the variable kinetics of the enzyme-substrate reaction; however, the amount of light emitted from fluorophores is highly consistent. The amount of light is directly proportional to the amount of protein on the membrane. This consistency means fluorescent detection can provide a truly quantitative analysis of the proteins in question.
AzureSpectra Fluorescent Secondary antibodies are labeled with fluorophores that emit light in visible and near-infrared wavelengths. The AzureSpectra 490, 550, 650, 700 and 800 secondary antibodies offer unparalleled sensitivity and performance for immunoblotting applications when used in conjunction with Azure Western blotting imaging systems.
| AzureSpectra | Excitation maximum (nm) | Emission Maximum (nm) |
|---|---|---|
| Dye-490 | 491 | 515 |
| Dye-550 | 551 | 565 |
| Dye-650 | 653 | 672 |
| Dye-IR700 | 690 | 709 |
| Dye-IR800 | 690 | 800 |
The Azure 600 Imager 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 3 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 beperformed – strengthening your data and your science.
| 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 |
Instructions for use, tips, troubleshooting and further information are available in the AzureSpectra AzureSpectra NIR Secondary Antibodies Product Insert instruction manual and Transition from Chemiluminescent to Fluorescent Western Blots instruction manual.
| Catalog Number | Description | Size |
|---|---|---|
| AC2206 | Goat-anti-rabbit 490 | 250uL at 1mg/mL |
| AC2207 | Goat-anti-mouse 490 | 250uL at 1mg/mL |
| AC2208 | Goat-anti-human 490 | 250uL at 1mg/mL |
| AC2209 | Goat-anti-chicken 490 | 250uL at 1mg/mL |
| AC2210 | Goat-anti-rat 490 | 250uL at 1mg/mL |
| AC2211 | Goat-anti-guinea pig 490 | 250uL at 1mg/mL |
| AC2217 | Donkey-anti-goat 490 | 250uL at 1mg/mL |
| Catalog Number | Description | Size |
|---|---|---|
| AC2158 | Goat-anti-rabbit 550 | 250uL at 1mg/mL |
| AC2159 | Goat-anti-mouse 550 | 250uL at 1mg/mL |
| AC2160 | Goat-anti-human 550 | 250uL at 1mg/mL |
| AC2161 | Goat-anti-chicken 550 | 250uL at 1mg/mL |
| AC2162 | Goat-anti-rat 550 | 250uL at 1mg/mL |
| AC2163 | Goat-anti-guinea pig 550 | 250uL at 1mg/mL |
| AC2164 | Donkey-anti-goat 550 | 250uL at 1mg/mL |
| Catalog Number | Description | Size |
|---|---|---|
| AC2165 | Goat-anti-rabbit 650 | 250uL at 1mg/mL |
| AC2166 | Goat-anti-mouse 650 | 250uL at 1mg/mL |
| AC2167 | Goat-anti-human 650 | 250uL at 1mg/mL |
| AC2168 | Goat-anti-chicken 650 | 250uL at 1mg/mL |
| AC2169 | Goat-anti-rat 650 | 250uL at 1mg/mL |
| AC2170 | Goat-anti-guinea pig 650 | 250uL at 1mg/mL |
| AC2171 | Donkey-anti-goat 650 | 250uL at 1mg/mL |
Due to low background autofluorescence in the near-infrared region, AzureSpectra 700 and 800 secondary antibodies can produce higher signal-to-noise ratios.
| Catalog Number | Description | Size |
|---|---|---|
| AC2128 | Goat-anti-rabbit 700 | 250uL at 1mg/mL |
| AC2129 | Goat-anti-mouse 700 | 250uL at 1mg/mL |
| AC2130 | Goat-anti-human 700 | 250uL at 1mg/mL |
| AC2131 | Goat-anti-chicken 700 | 250uL at 1mg/mL |
| AC2132 | Goat-anti-rat 700 | 250uL at 1mg/mL |
| AC2133 | Goat-anti-guinea pig 700 | 250uL at 1mg/mL |
| AC2156 | Donkey-anti-goat 700 | 250uL at 1mg/mL |
| Catalog Number | Description | Size |
|---|---|---|
| AC2134 | Goat-anti-rabbit 800 | 250uL at 1mg/mL |
| AC2135 | Goat-anti-mouse 800 | 250uL at 1mg/mL |
| AC2136 | Goat-anti-human 800 | 250uL at 1mg/mL |
| AC2137 | Goat-anti-chicken 800 | 250uL at 1mg/mL |
| AC2138 | Goat-anti-rat 800 | 250uL at 1mg/mL |
| AC2139 | Goat-anti-guinea pig 800 | 250uL at 1mg/mL |
| AC2157 | Donkey-anti-goat 800 | 250uL at 1mg/mL |
In fluorescent Western blotting, the secondary antibody is directly conjugated to a dye, which is excited by light. The emitted light is detected by a digital imager such as an Azure 400, rather than X-ray film (Figure 2) and digitized for data analysis. Multiple proteins can be detected simultaneously by using secondary antibodies conjugated to different dyes with non-overlapping spectral emissions.
The newer generation of imaging systems often contain sophisticated CCD cameras that exhibit a broader dynamic range than film, thus avoiding the signal saturation problems that limit the dynamic range of film. In addition, fluorescent dyes are relatively stable; blots can be archived and imaged months after the initial experiment as long as precautions are taken to avoid photo-bleaching of the fluorophores.
Azure imaging systems offer:
Multiplex fluorescence capabilities
Speed and sensitivity of X-ray film
Learn more about the Azure Imaging Systems, including full product specifications, user manuals and resources.
Fluorescent Western blots can be tricky. Don’t worry, we’ve got you covered. Azure’s Fluorescent Demo Kits contain everything you need to perform a single-color fluorescent Western using your own primary antibody and protein sample of your choice. Each kit contains enough material for 1 Western blot.
FREE WESTERN BLOT eBOOK
Able to detect up to 4 multiplexed fluorescent reactions per Western blot
Designed for multicolor
fluorescent Western blotting, with two NIR and 3 visible fluorescent channels
Three-color fluorescent detection for dyes in the visible range
Check out paper from the NIH that uses multiplex fluorescent Western blots to research implications for SARS-CoV-2 infection after repeated ethanol exposure.
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