Odyssey Chemifluorescent Substrates

  • Detect targets that do not work well with IRDye secondary antibodies
  • Combine chemiluminescence with the benefits of near-infrared fluorescence
  • Easy to incorporate into existing chemiluminescence detection protocols

Chemifluorescence: when IRDye labeled antibodies just won't do the job

Sometimes protein targets simply refuse to be detected with IRDye-labeled secondary antibodies. In these cases Odyssey Chemifluorescent Substrate is the solution.

With the Odyssey Chemifluorescent Substrate, you can still get the benefits of Odyssey near-infrared fluorescence detection, but you can use a conventional chemiluminescent protocol using an HRP-conjugated secondary antibody. The only change is the chemifluorescent substrate, which generates a precipitate that fluoresces and can be detected in the 700nm channel of Odyssey Imaging Systems.

Kit components

Odyssey Chemifluorescent Substrate Kit (Nitro)

  • 1 x 50 mL Odyssey Substrate Solution
  • 1 x 50 mL Odyssey Oxidant Solution
  • 10 Pre-cut Odyssey nitrocellulose membranes

Odyssey Chemifluorescent Substrate Kit (PVDF)

  • 1 x 50 mL Odyssey Substrate Solution
  • 1 x 50 mL Odyssey Oxidant Solution
  • 10 Pre-cut Immobilon®-FL PVDF Membranes

Procedure for chemifluorescent HRP detection

  1. Use the nitrocellulose membranes provided in the kit to perform Western blot detection. Process blot(s) with the same blocking buffers, antibody diluents, concentrations of primary and HRP-conjugated secondary antibodies, and wash buffers used for chemiluminescent detection.
  2. After the final wash step, rinse the membrane in detergent-free wash buffer (i.e. PBS or TBS) to remove residual wash buffer detergent. Allow the blot to remain in the final rinse solution until ready to begin detection with the Odyssey Chemifluorescent Substrate (within 30 minutes, to avoid degradation of the HRP enzyme).
  3. Prepare a Working Solution of Odyssey Chemifluorescent Substrate by mixing equal parts of Substrate Solution and Oxidant Solution. Prepare 0.1 mL Working Solution per cm2 of membrane (e.g. 5 - 6 mL per mini-blot). NOTE: Prepare the Working Solution at time of use; do not store.
  4. Remove blot from the final rinse solution (step 2), and incubate in Working Solution for at least 5 minutes, but no more than 10 minutes.
  5. Drain excess reagent. Cover blot and remove excess Working Solution. NOTE: For best results, place the blot inside a standard, non-glare, office sheet protector, roll out excess Working Solution, and make sure the top and bottom suffaces of the sheet protector are completely dry. Avoid direct contact of the Odyssey's silicone mat to the substrate.
  6. Image the blot on an Odyssey Imaging System, or any instrument capable of imaging blots at a wavelength of —700 nm.

Ordering information

Westburg offers this product in The Netherlands, Belgium, Luxembourg,
If you are not located in these countries please visit the LI-COR website to find your local distributor.
Cat no. Description
LI 928-30000 Odyssey Chemifluorescent Substrate (Nitro) kit
LI 928-30002 Odyssey Chemifluorescent Substrate (PVDF) kit
LI 928-30005 Odyssey Chemifluorescent Substrate Kit