Imaging Systems for Westerns: Chemiluminescence vs. Infrared Detection

Summary

Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging. Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum. Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal. A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.