Fluorescent western blot protocol- IRDye® secondary antibodies

Get your fluorescent western blot protocol. Learn how to use IRDye® ​secondaries, blocking, antibody dilutions, incubation times, washing steps and imaging to get clear bands in your fluorescent western blot.

Fluorescent western blot with secondary antibodies conjugated to IRDye® is quantitative and provides wider dynamic range than enzyme-based approaches, making it best option for quantifying relative protein abundance.

For guidance on sample preparation, running gels, and transferring protein from gel to membrane, see our general western blot protocol.

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Membrane blocking and probing with primary antibody

  1. Place membranes in Petri dishes and cover with high quality TBS-based blocking buffer (BB). Incubate for 1 hour.
  2. Dilute antibody in dilution buffer (50 % BB, 50% TBS 0.1% Tween 20 (TBST)).

    For each multi-tray well, 5 mL of antibody solution will be used. For petri dishes, 10 mL is required.
  3. Incubate membranes with 10 mL of the primary antibody solution on a rocking platform. Optimal dilution and incubation times should be determined using serial dilutions and a time course.

Washing the membranes and preparing the secondary antibody

  1. Discard the primary antibody solution and begin washing steps, keeping the membrane in the original Petri dish/wash tray. 
  2. Wash the membrane with TBST using 2 brief rinses, 5 mL each, followed by enough buffer to cover the membrane for 1 x 15-minute wash. Perform 3 subsequent 10-minute washes at room temperature.

    Place the wash trays and petri dishes on a rocker during the washes ensuring that the blots are washed separately.

  3. Between washes, dilute the secondary antibodies in antibody dilution buffer according to the WB experimental plan (see Table 1 below). Ensure that the secondary antibody is thoroughly mixed with the dilution buffer.

    All secondary antibodies are light sensitive and should be covered with aluminium foil or similar during incubation.

  4. Discard the TBST and add the required amount of secondary antibody. Incubate the membrane with the corresponding secondary on the rocker at room temperature for 1 hour and protected from light.
  5. After incubation, discard the secondary solution and repeat washing procedure in Step 2, keeping the membrane in the original Petri dish or wash tray.

Table 1: Secondary antibodies and dilutions

See other IRDye® products


  1. Clean the glass scanning bed with 70% ethanol using a cotton lint-free cloth.
  2. If scanning the membranes dry, place them between two sheets of filter paper and cover with foil and leave on the bench overnight. Place the dry membranes protein side down onto the glass scanning bed the silicon mat on top of the membrane to ensure the membranes lie flat against the glass. There is no need to roll.
  3. If scanning the membranes wet, place the membranes on the scanning bed protein side down and spray 5 mL of TBST across the scan area. Place a silicon mat on the membranes and use a roller to remove any bubbles.

    Scanning the membranes wet works well with histones.
  4. Close the equipment lid. Scan strips accordingly to equipment instructions. Remove the membranes from the scan bed and clean the scan bed with 70% ethanol using a cotton lint-free cloth.

>>Read next: Hints and tips for successful fluorescent western blot

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