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 System, using infrared fluorescence detection, reproducibly detected 1.2 pg of Tf, while only 4.9 -- 9.8 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).

Resources

• Protocols and Documents

  • One blot Western optimization using the MPX™ Blotting System
    LI-COR Biosciences (2009)

  • Good Westerns Gone Bad
    LI-COR Biosciences (2008)

  • Western Blotting Methods
    LI-COR Biosciences (2007)

  • Odyssey Western Blot Blocker Optimization
    LI-COR Biosciences (2009)

  • Protein Electrotransfer Methods and the Odyssey Infrared Imaging System
    LI-COR Biosciences (2009)

  • Odyssey Quick Card Reference for Western Blots
    LI-COR Biosciences (2008)

  • NewBlot™ Nitro Stripping Buffer for Nitrocellulose Membranes
    LI-COR Biosciences (2007)

  • NewBlot™ PVDF Stripping Buffer for PVDF Membranes
    LI-COR Biosciences (2007)

  • Ambroz, K. Improving Quantification Accuracy for Western Blots
    LI-COR Biosciences (2006)

  • Schutz-Geschwender, A et al. Quantitative, Two-Color Western Blot Detection with Infrared Fluorescence
    LI-COR Biosciences (2004)

  • See a complete list of pack inserts and protocols

• Webinars and Video Tutorials

  • "Chemi IR™ Detection of Western Blots on the Odyssey Infrared Imager"
    Shawn Mischnick, LI-COR Biosciences

  • “Odyssey Infrared Imaging System"
    Amy Geschwender, LI-COR Biosciences

  • “Quantitative, Multiplexed Western Blot Detection with Infrared Fluorescence”
    Amy Geschwender, LI-COR Biosciences

  • "Optimizing Signal Detection for Odyssey Western Blots"
    LI-COR Biosciences

  • Odyssey Power Users’ Guide
    LI-COR Biosciences

PubMed

Publishing with LI-COR data?

• Expression levels and extent of phosphorylation of cell signaling proteins

  Two independent fluorescent channels allow both total and phosphorylated target protein levels to be measured, even when the bands co-migrate.

  
  

  Markovic et al. used quantitative Western blotting to study how corticotropin-releasing hormone receptor-2β is involved in UCN-II activation of ERK1/2 and p38MAPK signaling. A time course of ERK1/2 and p38 MAPK activation by UCN-II was generated using two-color Western blotting. The contributions of β-arrestin1 and -2 in CRH-R2β desensitization and internalization were also assessed by RNAi . Reduced expression of each β-arrestin subtype in siRNA-transfected cells was confirmed by immunoblotting.

  
  

  Markovic, D et al.
  Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades.
  Mol Endocrinol. 22(3): 689-706 (2008)

  
  
• RNAi knockdown

  Ramsay et al. examined the specificity of KLK4 signaling via PAR-2, using siRNA to reduce expression of PAR-2. Three siRNA constructs were stably transfected into mouse PAR-2-LMF cells, and extent of knockdown was quantified by Odyssey Western blot. The best construct reduced PAR-2 levels by ~80%.

  
  

  The effect of PAR-2 knockdown on intracellular signaling was examined by assessing ERK1/2 activation in cells stably transfected with the siRNA construct. Quantitative Western blotting was again used. When treated with KLK4, transfected cells showed a ~70% reduction in ERK1/2 phosphorylation.

  
  

  Ramsay, AJ et al.
  Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression.
  J Biol Chem. 283(18): 12293-304 (2008)

  
  
• Activation of p53 (absolute protein quantification)

  Wang et al employed quantitative Western blots on Odyssey to quantify levels of p53, Hdm2 and Hdmx proteins in exponentially growing human cell lines (WS1, fibroblast cells; 184V, mammary epithelial cells) and tumorigenic cell lines (MCF7, mammary epithelial cells; U2OS, osteosarcoma cells). Using pure p53 protein and N-terminally tagged Hdm2 and Hdmx proteins, they performed absolute quantification of the number of molecules/cell.

  
  

  Wang, YV et al.
  Quantitative analyses reveal the importance of regulated Hdmx degradation for p53 activation.
  Proc Natl Acad Sci USA. 104(30): 12365-70 (2007)

  
  
• Validation of proteomic analysis

  Wishart el al. performed differential proteomics analysis to identify altered levels of protein expression in isolated synaptic preparations from WldS mice that displayed slowed neurodegeneration. Quantitative fluorescent Western blotting was used to confirm all candidates identified by 2D electrophoresis and mass spectrometry. Western blotting confirmed all mass spectrometry findings.

  Wishart, TM et al.
  Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.
  Mol Cell Proteomics. 6(8): 1318-30 (2007)

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