Comparison of chemiluminescence vs. Infrared techniques for detection of Fetuin-A in saliva

Abstract

The western blotting technique for transfer and detection of proteins, named following the discovery of southern and northern blotting for DNA-and RNA-blotting, respectively, has traditionally relied on the use of X-ray films to capture chemiluminescence. Recent advancements use super-cooled charge coupled devices (CCD) cameras to capture both chemiluminescence and fluorescence images, which exhibit a greater dynamic range compared to traditional X-ray film. Chemiluminescence detected by a CCD camera records photons and displays an image based on the amount of light generated as a result of a dynamic chemical reaction. Fluorescent detection with a CCD camera, on the other hand, is measured in a static state. Despite this advantage, researchers continue to widely 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 fluorophores in the infrared spectrum. Infrared imaging is static, has a wide linear range, high sensitivity, and reduced autofluorescence and light scatter. A distinct advantage of infrared imaging is the ability to detect two target proteins simultaneously on the same blot which increases accuracy of quantification and comparison, while minimizing the need for stripping and reprobing. Here, we compare the methodology for chemiluminescent (UVP BioChemi) and infrared (UVP Odyssey) detection of salivary total and phosphorylated fetuin-A, a multifunctional protein associated with cardio-metabolic risk, and discuss the advantages and disadvantages of these methodologies.