A Fundamental Change in Western Blot Analysis

Cat. No. LIC-9201-00

The Odyssey® Infrared Imaging System sweeps away the old paradigms of chemiluminescence and visible fluorescence, and introduces a new standard for Western blot analysis – direct infrared fluorescence detection. Infrared detection gives you the quantitative analysis and wide linear dynamic range that chemiluminescence can’t. Strong and weak bands on the same blot are accurately detected without the uncertainty and inconvenience of multiple exposures, and without spending time in the darkroom. The Odyssey System gives you clear, sharp, reproducible bands without fuzziness or “blowout”. Bands hidden by overexposure with chemiluminescence become clear when imaged with Odyssey

The Infrared Advantage:

In the visible wavelength range used by most fluorescent imaging systems, membranes and plastics produce high background due to light scattering and autofluorescence. This high background limits the sensitivity of visible fluorescent systems and makes it nearly impossible to detect low-abundance proteins at endogenous levels.

At the infrared wavelengths detected by Odyssey, both autofluorescence and light scatter are dramatically reduced. The result is the cleanest background, highest signal-to-noise ratios, and best detection sensitivity available with a fluorescent system.

Infrared dyes are the key to this performance advantage, and LI-COR Biosciences is a pioneer in the use of infrared fluorophores for imaging applications. We offer a unique family of IRDye® Infrared Dyes synthesized with reactive functionalities that enable easy covalent coupling to antibodies and other biomolecules. Additionally, the Odyssey system is compatible with a variety of other available fluorescent reagents. Please view our Fluorescent Reagent Compatibility Chart.

Features:

Accurate Quantification
Wide linear dynamic range
Through its innovative use of fluorescently labeled antibodies rather than enzyme labels, Odyssey provides a broad, linear dynamic range to accurately detect strong and weak bands on the same Western blot. In contrast, the dynamic enzymatic nature of chemiluminescence only allows you to capture a “snapshot” of the enzymatic reaction and is highly dependent on timing and exposure, limiting linear range and offering only qualitative or semi-quantitative results. The accuracy and linearity of Odyssey detection allow you to be confident about differences you see in protein levels, and your blots can be archived and imaged again months later, if needed.

Two-fold serial dilutions of Alexa® 680-labeled antibody (6 ng to 0.19 pg) were spotted on nitrocellulose and imaged in the 700 nm channel.

Multiplex Detection
Normalization increases quantification accuracy
The Odyssey system uses two infrared fluorescence channels for detection, enabling simultaneous two-color target analysis – an advantage that’s not available with chemiluminescent or radioactive methods. Two-color Western analysis makes normalization easy and eliminates error introduced by stripping and reprobing or by comparison of separate blots. Superior image clarity and detail makes it easier to detect subtle mobility shifts caused by protein modifications such as phosphorylation.

ERK1/2 and phospho-ERK were detected simultaneously in lysates of unstimulated and EGF-stimulated A431 cells. Two fold serial dilutions of lysate are shown. The single-color images can be overlaid to show both total ERK and phospho-ERK (yellow color indicates overlap of red and green signals). The mobility shift caused by phosphorylation can be seen in the EGF-stimulated lysate.

Direct Detection
No film, darkroom, or messy substrates
Odyssey is uniquely equipped with two infrared channels for direct fluorescence detection on membranes. With two detection channels, you can probe two separate targets in the same experiment. Quantification accuracy is increased when the second color is used for normalization.
Odyssey provides a flexible, multifunctional platform to accommodate a variety of applications, so you get results faster. One example is the In Cell Western assay, an immunocytochemical technique performed with cultured cells in microtiter plates. This assay uses target-specific antibodies to quantify protein levels in fixed cells. Accuracy, reproducibility, and throughput are all increased by eliminating time-consuming, error prone steps such as lysate preparation and gel electrophoresis.

Clear Data
No loss of weak bands due to overexposed bands

High Sensitivity
Equal to or better than chemiluminescence

Wide Range of Applications
Western blots, Coomassie® stained gels, cell-based assays, fluorescent gel-shift assays, tissue imaging, in vivo imaging, protein arrays, and more.

Coomassie-Stained SDS-PAGE Gel

Unstained BioRad Precision protein standards were loaded across a 10%SDS-PAGE gel, separated by electrophoresis and stained with Coomassie Blue.

In-Cell Western Assay
The unique advantages of infrared fluorescence allow In-Cell Westerns to provide extremely sensitive and quantitative analysis of cellular signaling pathways in cultured cells in a higher throughput manner. Use of infrared fluorescence reduces interference from cell, plate, and drug compound autofluorescence when compared to standard methods.

In-Cell Westerns simultaneously detect two targets at 700 and 800 nm using two spectrally distinct dyes. Separate lasers and fluorescence detectors are used for each dye and offer a wide linear detection range.

Odyssey MousePOD in vivo Imaging Accessory

• Fits over Odyssey® scanning surface
• Accommodates up to 3 mice
• Regulates air temperature
• Facilitates gas anesthesia

Infrared EMSA Analysis
Existing mobility shift assay protocols can be easily transformed into infrared assays by replacing the existing oligonucleotides with IRDye® end-labeled oligonucleotides. The binding conditions and electrophoresis conditions should remain the same as with any other EMSA detection method but may require some optimization.

Infrared AP-1 EMSA was used to visualize the changes in AP-1 binding following three different treatments of Hela cells. Hela, Hela 2 hour serum response, and Hela 4 hour serum response nuclear extracts were serially diluted and assayed under the same conditions using IRDye®700 end-labeled oligonucleotide duplex containing AP-1 binding sequence. Following electrophoresis the gel was imaged while remaining in the glass plates.

Protein Array Analysis

LI-COR now offers slide prep accessories and add-on software for protein array analysis on the Odyssey® Infrared Imaging System.

Specifications: