LI-COR Odyssey For High-Sensitivity Quantitative Western Blotting

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.

How does the Li-COR Odyssey Platform Work?

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.

Ideal Li-COR Applications: Quantitative Western Blots, Protein Gel Imaging, And In-Cell Western assays

In the LI-COR Odyssey infrared imaging system, the sensitive two-color near IR fluorescence detection allows simultaneous total and phosphorylated protein quantitation without stripping and reprobing the blots. We can also use Li-COR for gel imaging or in-gel Westerns for proteins that are too small, too large, or do not transfer well to membranes. Another assay where we can use LI-COR is the in-cell western used to detect and quantify proteins in cells fixed to microplates. Here, we follow the same steps as in western blotting except that lysate preparation, quantification, electrophoresis, and membrane transfer get eliminated. In-cell western assays can be used to study cell signaling and G-protein coupled receptor function, internalization, and recycling in neurology and oncology.

Key Li-COR Benefits: Superior Sensitivity, Quantification, and Linear Range

• LI-COR Odyssey near-IR protein quantification to detect strong and weak bands on a western blot is accurate 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

Why Choose NorthEast BioLab for LI-COR Quantitative Western Blots?

Our scientists, at NorthEast BioLab, develop reliable bioanalytical methods and perform your sample analysis assay for various stages of drug development. We handily carry out your protein quantification using western blots for pharmacodynamics studies in preclinical and clinical studies. We use LI-COR western blots to accurately detect cell proliferation, apoptosis, cell signaling, and various biomarkers, and quantitate proteins in a fuller, linear range as well as transfer images and data files to clients for convenient and safe long-term storage.