Anxious when it comes to performing fluorescent multiplex western blotting? Don’t worry, we have 12 tips that will make you a pro:

  1. Use primary antibodies from different host species (for example, mouse and rabbit). Antibodies produced from two closely related species such rat and mouse often give cross-reactivity, even when the antibodies are cross-adsorbed.
  2. Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity.
  3. Always optimize the detection of each target singly before simultaneous detection of multiple targets. As some primary antibodies may be nonspecific and yield multiple bands on a blot, a single target detection will help determine the banding pattern of each antibody prior to a multiplex experiment. Antibody concentrations should be optimized by incubating the membrane in several dilutions of each antibody. Select the dilution that yields the highest signal to background ratio.
  4. Detect the strongest target in the blue channel, the middle target in the green channel, and reserve the red channel for the weakest target. Most membranes show higher background with shorter wavelength excitation light.
  5. Use fluorophores conjugated to secondary antibodies with distinct spectra so they can be optically distinguished from each other to avoid cross-channel fluorescence (i.e., red, green, and blue).
  6. When adapting a chemiluminescent protocol to fluorescent detection, primary antibody concentrations may need to be increased two- to five-fold. Secondary antibody concentrations may also have to be optimized; a good starting point is a 1:5,000 dilution.
  7. In order to maximize the signal to background ratio, use a membrane with low auto-fluorescence, such as a low fluorescence PVDF membrane.
  8. For blocking buffer, use 0.5–5% casein and up to 5% nonfat dry milk or up to 3% BSA dissolved in TTBS. Particulates in the buffer can settle on membranes and create fluorescent artifacts; use only high-quality reagents and/or filter sterilize all buffers.
  9. Use blunt forceps to handle the membrane from the edge and avoid scratching or creasing the membrane, which can produce artifacts during fluorescent detection.
  10. Use pencil to mark membranes as many inks fluoresce.
  11. To avoid fluorescent bromophenol blue from interfering with your gel images, do one of the following: 1) ensure the dye front has migrated away from all samples, 2) cut off the portion of the gel containing the dye front, or 3) omit bromophenol blue from the sample buffer.
  12. It is not necessary to perform immunodetection in the dark as normal lighting will not significantly photobleach fluorescently labeled antibodies. However, store stocks of fluorescently labeled antibodies in the dark


Paul Z. Liu, Ph.D., is a senior scientist at Bio-Rad, a leading provider of Western blotting equipment. For more information please visit

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