[ABOVE] The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. IR Fluorescence: Lasers are used to excite 700 nm and 800 nm IRDye fluorophores for 2 channel detection. Fluorescent emission is then detected to generate an image of the sample. At these IR wavelengths, membrane autofluorescence is very low. This method directly detects the fluorescently labeled conjugates. Because the fluorescent signal is very stable, samples can be archived and re-imaged. Chemiluminescence: Luminol substrate is applied to the sample, and is oxidized by horseradish peroxidase (HRP) enzyme in a light-generating reaction. This method is indirect, because it relies on the enzymatic reaction to report the location of the HRP conjugate. Chemiluminescent signals are dynamic and time-dependent. Light emission is initially strong, but diminishes over time.

Molecular weight markers can be visualized at 700 nm
on chemiluminescent Western blots

[LEFT] NIH/3T3 cell lysates were separated and transferred to a membrane. Odyssey Protein MW Markers (P/N 928-40001, 928-40000) (which are labeled with 700 nm IR fluorescent dye) were included in the left lane. Akt was then detected, using appropriate antibodies and ECL Plus™ (GE Healthcare) chemiluminescent substrate.

Akt signal was acquired with the Odyssey Fc System in the chemiluminescence channel (white bands) and 700 nm fluorescence channel (MW marker; red bands). The two signals were then overlayed in Image Studio software, so that both signals could be visualized simultaneously.