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ODYSSEY® Fc IMAGER
Applications for the
Odyssey Fc Imaging System
QUANTITATIVE IR WESTERN BLOT
Application Overview
Quantitative IR Western Blotting
Infrared fluorescent detection is a direct, nonenzymatic approach to Western blotting that uses secondary antibodies labeled with IRDye® infrared dyes (see "Why Infrared Fluorescence?" below). This method improves quantitative accuracy and reproducibility, and facilitates multiplexing. The stable fluorescent signal can be detected immediately, or after extended storage.
[ABOVE] The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. IR Fluorescence: Lasers are used to excite 700 nm and 800 nm IRDye fluorophores for 2 channel detection. Fluorescent emission is then detected to generate an image of the sample. At these IR wavelengths, membrane autofluorescence is very low. This method directly detects the fluorescently labeled conjugates. Because the fluorescent signal is very stable, samples can be archived and re-imaged.
Wide Dynamic Range for Accurate Quantification
The unusually wide dynamic range allows precise quantification of both strong and weak bonds, to more accurately reflect protein levels. Serial dilutions of purified transferrin (10 ng to 1.2 pg) were detected with rabbit anti-transferrin and IRDye® 800CW secondary antibody. Acquisition time with the Odyssey Fc Imager was 2 minutes. Triplicated blots were performed
Fluorescent signals on the triplicate blots were quantified with the Image Studio software. Errors bars are shown, but are very small. Results were linear across the entire range tested (4,000-fold: 3.6 orders of magnitude).
LI-COR Biosciences provides protocols, application notes, helpful tips and references to help you produce high-quality Western blotting results. For more information, visit biosupport.licor.com.
Quantification with Confidence
Through the innovative use of infrared fluorescent antibody conjugates, the Odyssey® Fc Imager provides a broad, linear dynamic range to accurately detect both strong and weak bands on the same Western blot. In contrast, the dynamic enzymatic nature of chemiluminescence allows you to capture only a "snapshot" of the enzymatic reaction and is highly dependent on timing and exposure.
The accuracy and linearity of fluorescence detection on the Odyssey Fc System allow you to feel confident about differences you see in protein levels. And your near-infrared fluorescent blots can be archived then imaged, and quantified again months later, if needed.
Why Infrared Fluorescence?
Unique to all Odyssey® platforms is the use of infrared laser excitation that out-performs visible fluorescence systems. In the visible wavelength range used by most fluorescence imagers, membranes and plastics produce high background due to light scattering and autofluorescence. This limits the sensitivity of visible fluorescence systems and makes it difficult to detect low-abundance proteins at endogenous levels without saturation of stronger bands.
Nitrocellulose and PVDF membranes were imaged with the Odyssey Infrared Imaging System at an Intensity = 5 for both 700 and 800 nm wavelengths. The same membranes were scanned at a 532 nm and 635 nm wavelength with a PMT = 500 on a GenePix® 4100A (Molecular Devices). Autofluorescence was much lower at infrared wavelengths.
