Fluorescent Western Blot Tips from Antibody Experts

Fluorescent western blotting can be frustrating work, especially if the application needs optimization or if the technique has never been used before. While running a fluorescent western blot there are several steps where potential problems can occur and affect the final outcome. Choosing the right antibodies and using them appropriately is especially important. Here we provide tips and tricks from our antibody R&D scientists to help get a fluorescent western blot right the first time.

Tips for steps of the fluorescent western blot workflow


  • Bromophenol blue is a common dye used in sample buffer as a dye front. It allows you to see the samples moving through the SDS-Page. Bromophenol blue also has a tendency to auto-fluoresce. To prevent this auto-fluorescence, make sure to either run the dye front off the gel or cut it off before transfer.


  • When moving the membrane use clean forceps; dirt from unclean forceps or fingerprints will fluoresce.
  • Any buffers (transfer, blocking, wash) that come in contact with the membrane should be filter-sterilized. This will help to avoid aggregates on the membrane that may become fluorescent artifacts.
  • Wipe all blot containers with 100% methanol before each use to further prevent the possibility of artifacts.
  • If marking the membranes do so with pencil. Ink will auto-fluoresce.

Probing the membrane

  • Perform probing steps using a primary antibody that has been functionally tested for western blotting applications using a concentration recommended by the manufacturer or prior empirical testing.
  • Following the addition of the fluorescent secondary antibody, cover the blot container with aluminum foil to protect the fluorophore from light. While our Alexa Fluor and Alexa Fluor Plus Secondary Antibodies are photostable, taking extra precaution to protect them from light will ensure light exposure is not a contributing factor to a poor looking fluorescent blot.
  • See “Multiplexing” below for additional information about primary and secondary antibody selection.

Blot imaging

  • Clean surface of imager with alcohol based cleaner before imaging to ensure a clear image.


  • Use primary antibodies from different species to prevent respective fluorescent secondary antibodies from binding to more than one primary antibody, which would result in a failure to optically differentiate between the multiple targets.
  • For best results, use a cross-adsorbed secondary antibodies to reduce the chance of cross-reactivity. A cross-adsorbed antibody is one that has gone through additional purification steps to filter out nonspecific-binding secondary antibodies. What is left is a highly specific secondary antibody that will minimize primary antibody species cross-reactivity and overall background.
  • Use fluorophore conjugates with optically distinct spectra to avoid cross-channel bleed through. To ensure that there is no overlap in the fluorophore conjugates use the Fluorescence SpectraViewer.
  • Use the 800 nm channel for detection of less abundant proteins or weak targets. Use the 680 nm channel for more abundant proteins or strong targets.