LI-COR Odyssey For High-Sensitivity Western Blot Analysis
LI-COR Odyssey is a flexible, IR laser-based instrument that offers enhanced data quality and sustained sensitivity and can readily detect even subtle mobility shifts due to protein modifications. It uses infrared fluorescent (IR fluorescent) antibody conjugates to identify strong and weak bands on a western blot. LI-COR adopts a simultaneous ratiometric approach with its 700 and 800 nm infrared (IR) fluorescent detection channels that facilitate a two-color target analysis and normalization.
The platform facilitates protein quantification in a wide linear range and offers flexibility for the blot processing and storage as the signal from the IR fluorescent dyes is robust. LI-COR Odyssey facilitates both chemiluminescent and IR fluorescent protein detection without film usage. This elimination allows flexibility, data archiving, darkroom and developer cost savings, and environmental friendliness.
Key Li-COR benefits include superior sensitivity, quantification, and linear range –
- LI-COR Odyssey near-IR protein quantification discern strong and weak bands on a western blot accurately because fluorescent detection is static, unlike the dynamic light generation in chemiluminescence
- The longer wavelengths of near-IR fluorescent detection result in reduced autofluorescence of membrane surfaces and biomolecules, resulting in lower signal-to-noise ratio and increased sensitivity with NIR fluorophores
- NIR allows a broader, linear detection range such that protein concentrations falling within the detection limits of the instrument are visible in the scanned image
- LI-COR Odyssey compares well in chemiluminescence scenarios, wherein optimization of exposure times for high protein concentrations leads to undetectable/unquantifiable bands for lower protein concentrations and vice-versa
Li-COR Odyssey: Near-Infrared Imaging for Western Blot Bands
Here’s a comprehensive Li-COR Odyssey protocol with some steps relevant only to appropriate protein assays, such as western blots, in-gel westerns, or in-cell westerns
- Perform polyacrylamide gel electrophoresis on protein samples
- Transfer the proteins to a nitrocellulose or polyvinylidene membrane
- Incubate membrane with primary antibodies and limit non-specific binding
- Incubate Near-IR (NIR) fluorescent secondary antibodies with the membrane to bind these with the primary antibodies
- Identify and quantify protein via ratiometric detection by the Li-COR Odyssey infrared imaging system after washing away unbound antibodies
In the Li-COR Odyssey platform, we use one of the two IR detection channels (700 and 800 nm) for our protein of interest and the other channel for a housekeeping protein. Afterward, we achieve accurate quantitation by normalizing the signal intensities from the two proteins. The technology can also be used to bridge enhanced chemiluminescence (ECL) and IR fluorescence imaging. In this case, we use appropriate near-IR fluorescent antibodies directed against horseradish peroxidase (HRP)-conjugated antibodies already bound to either primary antibodies or immobilized target proteins.