NIR fluorescence in the FUSION-FX7 SPECTRA system
Usually, the Western Blot Chemiluminescence detection clearly outperforms fluorescence detection concerning parameters such as detection limit or signal dynamics. This is particularly true for our high-performance CCD imagers of the FUSION family.
Nonetheless, general interest in detecting fluorescence has increased particularly for Western blot applications. So it may be desirable under certain circumstances to use the same label in various applications (eg eGFP in plant cells and also on the fluorescent Western blot). In addition, multiple labeling with various secondary antibodies without stripping - e.g. for the purpose of standardization with housekeeping genes - could be an interesting option.
In the past, FUSION imagers already could be equipped with powerful modules for the UV fluorescence and for the RGB-visible fluorescence.
In chemiluminescence detection, detectable light is generated at a coupled secondary antibody by enzymatic reaction of a substrate. In fluorescence measurement there is a light-source in the measuring field (laser, LED, UV), by which the coupled fluorophore is excited. By selection of suitable emission filters, the light of the excitation source can indeed be filtered out almost completely. However, there are still factors which could considerably influence the overall performance in fluorescence detection. One of the most important factors is non-specific autofluorescence of other substances in the sample like proteins, lipids, water, etc..
By effective choice of low fluorescence membranes and optimization of blotting parameters (concentration of secondary antibody, washing solutions, blocking agents), nonspecific autofluorescence can be decreased significantly. However, usually there still remains a higher non-specific background in comparison to chemiluminescence. This is the main target of our new IR SPECTRA technology. At the longwave end of the visible spectrum (700 to 800 nm), the extinction coefficients of many proteins decrease. The extinction coefficients of solvents (such as water) on the other hand increase only at higher wavelengths. Compared to an excitation within ultraviolet or in visible (RGB-) range, excitation in the near infrared (NIR) region may decrease nonspecific autofluorescence significantly, thus improving the signal-to-noise ratio and hence the limits of detection considerably.
Any fluorescence Western blot application typically suffering from autofluorescence, or with the need to perform a duplex application with the lowest possible cross-talk between the channels will benefit from this new technology.
In particular, these applications include:
· Western blot of total cell lysates
· Protein binding studies
· Cell-Signaling analysis
· Experiments in protein-protein interactions
· Phosphorylation studies
· generally all Western blot applications where two different proteins of the same size are to be differentiated
The NIR SPECTRA FUSION module is the latest upgrade to our proven FUSION multi-application imagers that now also offer detection of NIR fluorescence in addition to the detection of chemiluminescence, UV fluorescence and RGB (VIS) fluorescence. The NIR spectra module is easy to upgrade and is completed by appropriate emission filters. Thus, the FUSION multi-application imager now offers the complete spectral range from the ultraviolet to the infrared wavelength.