photo of gel with multicolor fluorescent bands 

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 Invitrogen 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.

  • Simple, quantitative western blots
  • Uses existing benchtop equipment
  • Wide linear dynamic range
  • Multiplexing capability

Choose your imaging platform:

photo of gel with red-fluorescent bands 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.

WesternDot 625 conjugates can be imaged on transilluminator systems such as those designed for ethidium bromide or SYBR Green gel stains.

ConjugateWesternDot 625
Ex/Em405-485/625 nm
Donkey anti-mouseW10805
Donkey anti-rabbitW10806
Donkey anti-goatW10807
Goat anti-mouseW10808
Goat anti-rabbitW10809
Anti-mCherry rat monoclonalW10829
Anti-His Tag mouse monoclonalW10833
black and white photo of gel from gel imager 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.

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 iBright Imaging Systems.

ConjugateWesternDot 585WesternDot 625WesternDot 655WesternDot 800
Ex/Em405-485/585 nm405-485/625 nm405–485/655 nm405–485/800 nm
Donkey anti-mouseW10800W10805W10810W10823
Donkey anti-rabbitW10801W10806W10811W10824
Donkey anti-goatW10802W10807W10812W10825
Goat anti-mouseW10803W10808W10813W10815
Goat anti-rabbitW10804W10809W10814W10816
Goat anti-rat  W10819W10821
Goat anti-chicken  W10820W10822
Anti-mCherry rat monoclonalW10828W10829W10830W10831
Anti-His-Tag mouse monoclonalW10832W10833W10834W10835
composite photo of fluorescent bands from four gels 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.

Best results for multiplex imaging are achieved using probes with broad spectral separation and a multiplexing imaging platform. 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 iBright Imaging Systems.

ConjugateWesternDot 585WesternDot 655WesternDot 800
Ex/Em405-485/585nm405–485/655 nm405–485/800 nm
Donkey anti-mouseW10800W10810W10823
Donkey anti-rabbitW10801W10811W10824
Donkey anti-goatW10802W10812W10825
Goat anti-mouseW10803W10813W10815
Goat anti-rabbitW10804W10814W10816
Goat anti-rat W10819W10821
Goat anti-chicken W10820W10822
Anti-mCherry rat monoclonalW10828W10830W10831
Anti-His-Tag mouse monoclonalW10832W10834W10835
Transilluminator
photo of gel with red-fluorescent bands 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.

WesternDot 625 conjugates can be imaged on transilluminator systems such as those designed for ethidium bromide or SYBR Green gel stains.

ConjugateWesternDot 625
Ex/Em405-485/625 nm
Donkey anti-mouseW10805
Donkey anti-rabbitW10806
Donkey anti-goatW10807
Goat anti-mouseW10808
Goat anti-rabbitW10809
Anti-mCherry rat monoclonalW10829
Anti-His Tag mouse monoclonalW10833
Gel Imager
black and white photo of gel from gel imager 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.

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 iBright Imaging Systems.

ConjugateWesternDot 585WesternDot 625WesternDot 655WesternDot 800
Ex/Em405-485/585 nm405-485/625 nm405–485/655 nm405–485/800 nm
Donkey anti-mouseW10800W10805W10810W10823
Donkey anti-rabbitW10801W10806W10811W10824
Donkey anti-goatW10802W10807W10812W10825
Goat anti-mouseW10803W10808W10813W10815
Goat anti-rabbitW10804W10809W10814W10816
Goat anti-rat  W10819W10821
Goat anti-chicken  W10820W10822
Anti-mCherry rat monoclonalW10828W10829W10830W10831
Anti-His-Tag mouse monoclonalW10832W10833W10834W10835
Multiplex Gel Imager
composite photo of fluorescent bands from four gels 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.

Best results for multiplex imaging are achieved using probes with broad spectral separation and a multiplexing imaging platform. 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 iBright Imaging Systems.

ConjugateWesternDot 585WesternDot 655WesternDot 800
Ex/Em405-485/585nm405–485/655 nm405–485/800 nm
Donkey anti-mouseW10800W10810W10823
Donkey anti-rabbitW10801W10811W10824
Donkey anti-goatW10802W10812W10825
Goat anti-mouseW10803W10813W10815
Goat anti-rabbitW10804W10814W10816
Goat anti-rat W10819W10821
Goat anti-chicken W10820W10822
Anti-mCherry rat monoclonalW10828W10830W10831
Anti-His-Tag mouse monoclonalW10832W10834W10835

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).

Direct detection of mCherry and His-tagged proteins

We've developed primary antibody conjugates of WesternDot probes for the direct detection of mCherry and His-tagged proteins. These intensely fluorescent WesternDot primary antibodies reliably and specifically detect the protein target in western blot applications without the need for a secondary antibody. Blots can be  imaged immediately after primary antibody incubation and wash steps or stored for later.

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.

graphs showing dynamic range of signal

Functional multiplexing

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.

Resources

For Research Use Only. Not for use in diagnostic procedures.