Western Blot analysis is a well-established method for protein detection and widely used in many laboratories. Traditional Western Blot analysis is time sensitive with respect to membrane exposure to the substrate. This makes quantification and reproducibility with chemiluminescence a problem. The Li-Cor Odyssey Infrared Imaging System overcomes these problems by eliminating the need for a substrate. This results in reproducible results, flexibilty on scanning the membrane, and easy quantification. Two separate lasers and detectors simultaneously and independently scan the fluorescent signal. The dual channels allow for simultaneous detection of two or more separate target samples on the same membrane. Since detection occurs directly on the membrane, the dark room and x-ray film are obsolete, saving time and money. In addition, when protected from light, membranes are stable for up to a year or more and can be re-scanned without significant degradation.
The Odyssey system makes use of an infrared detection method which effectively eliminates background fluorescence, resulting in clear, sharp bands. Strong and weak bands on the same blot are accurately measured, since the direct infrared detection allows for a wider linear range. Bands that are hidden in overexposed areas with chemiluminescence suddenly become clear when imaged with the Odyssey System. The scans are saved to a 90-Gigabyte internal hard drive and can be accessed via the computer that comes with the system or through a password protected internet IP.
Of course, to make use of the infrared detection system, specialized secondary antibodies are required. The Alexa Fluor® 680-labeled secondary antibodies can be purchased from Molecular Probes and the LI-COR IRDye™ 800-labeled secondary antibodies can be purchased from Rockland Immunochemicals. Also, a labeling kit can be purchased to perform the conjugation of the fluorophore to the primary or secondary antibody of choice in-house.
Some of the other benefits of the Odyssey include: extensive on-site training and set-up, helpful and knowledgeable technical support, relatively small footprint, and user-friendly software.
Although the Odyssey’s main selling feature is the use in Western Blot analysis, it is also used for In-Cell Westerns, two color EMSA, two color Westerns, two color in-gel Westerns, two color Northern blots, protein phosphorylation, protein gel documentation, 2D-gel documentation and in some instances southern blot analysis. Our labs have so far used it exclusively for western blot analysis and find the Odyssey to be a great tool for research.
Todd O’Buckley
Lab Manager
UNC Chapel Hill Center for Alcohol Studies