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ODYSSEY® Sa IMAGER
Applications for the
Odyssey Sa Imaging System
QUANTITATIVE WESTERN BLOTTING
Application Overview
[ABOVE] Figure 1.
Induction of JNK phosphorylation in response to LPS treatment. Phospho-JNK is shown in green, total JNK in red. Yellow indicates overlap in pseudocolor overlay.
Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008)
For detection, replace your HRP-conjugated antibody with secondary antibodies labeled with IRDye infrared dyes.
Two protein targets can be detected in separate fluorescent channels, even if the bands co-migrate
Better image clarity for sharper, more detailed bands
Detect subtle mobility shifts caused by protein modifications such as phosphorylation
[ABOVE] Figure 2. Serial dilutions (10 ng to < 1 pg) of purified human transferrin (Tf) were used to assess Western sensitivity. The Odyssey Classic, using infrared fluorescence detection, reproducibly detected 1.2 pg of Tf, while only 4.9 -- 9.5 pg was detected with chemiluminescence. Infrared detection sensitivity was approximately 200-fold greater than previous studies with visible fluorophores (Cy®3, Cy®5, or FITC).
Advantages
Figure 1
Odyssey detection offers many general advantages over chemiluminescence:
Get out of the darkroom
Place instrument on the bench in your lab
No waiting for the darkroom while your signal fades
Fluorescent signal is indefinitely stable
Image your blot at your own convenience
Environmentally friendly
No film, developer, or water waste hassles
Sharper bands
No fuzziness or “blowout”
Capture faint and strong bands in the same image
Time and cost savings.
No substrates or film
No exposure times to optimize
Wide Linear Dynamic Range
Odyssey fluorescent detection provides the widest linear dynamic range, making
protein quantification simpler and more accurate
Western blot detection of Hsp70 is reported to be linear over 4.3 orders of magnitude,
from 5 pg to 100 ng1 (Figure 1)Direct comparison demonstrates that Odyssey detection provides a greater dynamic range
than ECL chemiluminescence2 (Figure 1)Compare against a concentration standard for absolute quantification2
Gather all the information you need from a single blot or image
[LEFT] Figure 1. Relationship between protein concentration of the hsp70 calibrant and fluorescence intensity. (a) Various concentrations of a hsp70 calibrant were assessed via western blotting. (b) There was a linear relationship between protein concentration and signal intensity across the full range from 5 pg to 100 ng – a range of 20,000 fold or 4.3 orders of magnitude.
Reprinted with permission from Bromage et. al. Marine Ecol. Prog. Ser. 376:123-132 (2009).
[ABOVE] Figure 2. Comparison of detection methods for Western blot analysis. Pure recombinant P53, Hdm2, and Hdmx protein samples of known concentration were serially diluted with 100 ng/ml BSA and run in duplicate on two gels, followed by Western blot analysis. Proteins were detected by using either LI-COR or standard ECL. Signal intensities from each Western blot were quantified by using either the LI-COR/Odyssey infrared image system or, for ECL, scanning the developed films, followed by analysis with Un-Scan-It software. Signal intensities were plotted against corresponding protein concentrations.
Reprinted with permission from: Wang, YV et al. Proc Natl Acad Sci USA. 104 (30): 12365-70 (2007) Copyright (2007) National Academy of Sciences, U.S.A.
Multiplexing
The Odyssey offers two independent channels (colors) for normalization or loading controls, so that two protein targets can be detected in separate fluorescent channels, even if the bands co-migrate.
[LEFT] Figure 1. Total EGF receptor (EGFR) and phospho-EGFR were detected simultaneously in lysates of unstimulated and EGF-stimulated A431 cells. Two fold serial dilutions of lysate were loaded. The single-color images can be merged to show both total and phospho-EGFR (yellow indicates overlap of red and green signals). The mobility shift caused by phosphorylation can be seen in the EGF-stimulated lysate (red signal above yellow band).
Support Materials
Need help identifying a primary antibody?
Protocols and Documents
One blot Western optimization using the MPX™ Blotting System
Protein Electrotransfer Methods and the Odyssey Infrared Imaging System
NewBlot™ Nitro Stripping Buffer for Nitrocellulose Membranes
Papers
Schutz-Geschwender, A et al. Quantitative, Two-Color Western Blot Detection with Infrared Fluorescence
Ambroz, K. Improving Quantification Accuracy for Western Blots
See a complete list of protocols and support documents
Webinars and Video Tutorials
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