Quantitative Westerns

NIR-labeled secondary antibodies yield a much wider linear detection range than chemiluminescence on Western blots (Figure 1). 

Chemiluminescence Linear Range: 250-fold   Odyssey Linear Range: >4000-fold

Figure 1. A dot blot assay was used to compare the linear ranges of chemiluminescent and infrared fluorescent detection. Dilutions of mouse antibody were spotted as antigen and detected with HRP- or infrared-labeled goat anti-mouse antibodies. Chemiluminescent data were collected using ECL substrate and a CCD camera with varying exposures; the infrared image was obtained in a single scan with the Odyssey system. For a 30 minute chemiluminescent exposure, the data set was linear over a 250-fold range. In contrast, infrared detection displayed a quantitative linear range of > 4000 fold (3.6 orders of magnitude).

The practical result is illustrated in Figure 2. With chemiluminescence, optimizing exposure times for low protein concentrations may make bands with high protein concentrations unquantifiable (Figure 2E).  Similarly, optimizing for high protein concentrations may make bands with low protein concentrations undetectable (Figure 2B). When visualizing uncharacterized proteins, a given protein may not be detected at all using chemiluminescence, simply because the exposure was not optimal. With NIR detection, the wider linear detection range and static nature of fluorescence detection means that all protein concentrations within the detection limits of the instrument are visible in the scanned image and can be quantified.

Figure 2.  Near-infrared versus chemiluminescent detection of transferrin at concentrations of 1000 pg, 32 pg and 1 pg on nitrocellulose membrane. (A) Odyssey Infrared Imaging System, 800 nm scan, with an intensity setting of 4.  Chemiluminescent blot detected after (B) 30 seconds, (C) 1 minute, (D) 5 minutes, and (E) 10 minutes of exposure to film.