WesternDot™ Fluorescent Immunodetection Kits
Fluorescence detection enables quantitative, multiplex analysis of western blots right at your bench—without the need for ECL optimization, film, or a darkroom. Long-wavelength reporters like WesternDot® antibodies are detected on membranes with high sensitivity and minimal background signal or scatter. Detect both strong and weak signals at the same time with a >4,000-fold linear dynamic range. With an appropriate reader you can multiplex up to three probes on the same blot, providing an extra level of precision and biological context for your measurements.
Choose your imaging platform:
|The standard benchtop transilluminator you used for ethidium bromide or SYBR® Green detection is an ideal imaging system for simple fluorescent western blots. WesternDot® 625 conjugates exhibit a robust fluorescent signal with most transilluminator wavelengths. A standard gel imager with an ethidium bromide filter is an ideal starting point for simple WesternDot® probe detection. Place your blot face down on the stage for the best signal, and for rapid documentation you can even capture the result on a smart phone.|
|You can obtain precise, quantitative data from your WesternDot® gel blots using either a camera-based or scanner-based gel imaging system. Select the right wavelength for your imager and WesternDot® conjugate so that you maximize signal and minimize background. With an appropriate detector your blots will provide up to 4 orders of magnitude of linear dynamic range. That’s about 16 times more than chemiluminescent detection, with lower cost and a vastly simpler workflow. Use any of the WesternDot® conjugates for robust quantitative results.|
|With a multi-wavelength imaging system you can take advantage of the full power of WesternDot® conjugate detection. Use the multiplexing capability to monitor housekeeping proteins, correlate different protein concentrations, or simply run reference standards. No need for duplicate blots or reprobing—you can directly compare band intensities for multiple proteins on a single blot. Combine WesternDot® 585 conjugates with WesternDot® 655 and 800 conjugates for the best spectral separation and multicolor detection. The wide range of available conjugates offers you the flexibility you need.|
WesternDot® 625 conjugates can be imaged on transilluminator systems such as those designed for ethidium bromide or SYBR® Green gel stains.
Both camera-based and scanner-based gel imaging systems can be used to image and quantify single-color WesternDot® conjugates. Determine the wavelength capability of your imager and match it to an appropriate WesternDot® conjugate. Find probes compatible with LI-COR® imaging systems.
|Conjugate||WesternDot® 585||WesternDot® 625||WesternDot® 655||WesternDot® 800|
|Ex/Em||405-485/625 nm||405-485/625 nm||405–485/655 nm||405–485/800 nm|
Best results for multiplex imaging are achieved using probes with with broad spectral separation and an imaging platform such as Azure™ c500 or Fuji. Any combination of WesternDot® 585, 655, and 800 will give you multicolor detection without the need to strip and re-probe your blot. Find probes compatible with LI-COR® imaging systems.
|Conjugate||WesternDot® 585||WesternDot® 655||WesternDot® 800|
|Ex/Em||405-485/585nm||405–485/655 nm||405–485/800 nm|
Streamlined fluorescence workflow
Follow your standard blotting procedure to optimize binding and minimize background. WesternDot® conjugates are compatible with standard membranes and buffers, so your transfer process, blocking reaction, primary antibody incubation, and wash steps are the same as for ECL—the streamlined workflow and improved quantitation comes in the detection stage.
Now in place of your HRP- or AP-conjugated secondary antibody, use a WesternDot® antibody conjugate. After a standard 15-minute incubation period, wash the membrane and you’re ready to read.
No trip to the darkroom, no substrate, no film, no time sensitivity, and no development. Just place your blot on the trans-illuminator, scanner, or reader. You already have higher information content available to you (and you obtained it with less processing time).
Improved linear dynamic range
Long-wavelength WesternDot® probes allow signal collection beyond the range of typical membrane autofluorescence to provide exceptional signal-to-noise ratios. Unlike ECL, your signal depends on neither enzyme kinetics nor film response, so the readout is more stable, robust, and reproducible. The linear dynamic range of your detector will enable visualization of both strong and weak bands in the same blot.
With multiple fluorescence detection channels you can save both time and precious sample by detecting multiple proteins in the same assay. No need to strip and reprobe, or run a second blot. You can also normalize your intensity values to a control protein to correct for any inaccuracies in loading. For the most compelling results, you can monitor multiple proteins in the same experiment.