Multiplexing in fluorescent western blotting

Learn about the advantages of detecting multiple targets on the same blot at the same time with fluorescent western blotting.

Multiplexing in fluorescent western blotting allows you to:

  1. Quantify relative protein abundance
    Compare abundance of one protein over another. For instance, you can compare the abundance of a phosphorylated form of your protein of interest to the total amount of protein.
  2. Normalize in the same gel
    Do normalization of band intensity with an internal control in the same blot, without the inconveniences of stripping and reprobing again.

Why is quantification and multiplexing possible with fluorescent WB?

  • Fluorescent applications are more quantitative than enzyme-based approaches (eg HRP) making normalization against an internal control (eg housekeeping gene) easier and more accurate.
  • Fluorescent western blotting provides the widest dynamic range so both low- and high-abundant proteins can be detected in the same experiment.

For more the benefits of using fluorescence in WB see comparison to chemiluminescence here.

For fluorescent western blot, we recommend using our IRDye® secondaries:

  • Cross-adsorbed to minimize species cross-reactivity.
  • Have non-overlapping emission spectra to avoid cross-channel fluorescence.
  • Optimized for fluorescent western blot and tested in more than 600 different protein targets.


Use the green channel (IRDye® 800) to detect your weakest target and the red channel (IRDye® 680) to detect the strongest target.

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