NIR Fluorescence vs. Chemiluminescent Detection

Imaging systems used for Western blot analysis are often described as ‘quantitative’.  While it is true that the instrumentation used to image the blots does acquire signal in a quantitative way, data produced by these systems can only be quantitative if the signal generated by the chemistry is proportional to the amount of sample on the blot in a linear fashion. In other words, a system is quantitative if, when the amount of protein doubles on a blot, the signal generated by the detection system increases proportionally. 

Chemiluminescent detection has been the most popular protein detection method in Western blotting applications due to its sensitivity.  Chemiluminescent detection relies on an enzymatic reaction that produces light, which is detected by a CCD camera or imaged on film. The enzymatic reaction used to produce the light is dynamic, constantly changing over time. Some samples produce bright light for a short time, and others produced comparatively dim light, but for a long period of time. Therefore, images must be collected at an optimized time. This time-dependence of signal compromises quantification and accuracy.

Fluorescent detection, by comparison, is static. Light produced from the excitation of a fluorescent dye can be compared to a light bulb. When a fluorescent dye is excited, or ‘on’, the amount of light produced is constant. This makes fluorescent detection a more precise and accurate measure of the differences in signal produced by labeled antibodies bound to proteins on a Western blot. Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes is measured in a static state compared to chemiluminescence where light is measured in a dynamic state.

Secondary antibodies labeled with visible fluorophores have been available for Western blotting, however, their performance has been poor.  Near-infrared (NIR) fluorophores, such as IRDyes, provide excellent sensitivity with all the advantages of fluorescent detection. NIR detection is more sensitive than visible fluorescence and equal to or more sensitive than chemiluminescent detection (chemiluminescent detection sensitivity depends on which substrate and detection method are used).  At longer wavelengths, membrane surfaces and biomolecules exhibit greatly reduced autofluorescence, resulting in lower background and enhanced sensitivity when NIR fluorophores are used for detection. Untreated nitrocellulose and PVDF membranes have much lower autofluorescence when scanned in the NIR than in the visible range of the spectrum (Figure 1). As a result, there is a dramatic decrease in membrane associated background in the NIR. The reduction of background using NIR detection directly addresses the primary caveat of membrane-based protein detection with either chemiluminescence or visible fluorophores – low signal-to-noise ratio. NIR detection dramatically increases the signal-to-noise ratio for membrane-based applications.

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Figure 1.  Nitrocellulose and PVDF membrane were scanned on 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).