Fluorescent Western Blotting
Fluorescent blotting applications differ from other detection systems in that the signal is not a product of an enzyme reaction, but rather a transient light emission resulting from the excitation and subsequent release of photons as the excited molecule returns back to its normal state. This is in contrast to enzyme-substrate systems that produce a colored product or light emission as a result of an chemical reaction. This allows optimized fluorescent applications to be more quantitative than enzyme systems.
Similar to enzyme reactions, fluorescent reagents must be optimized for optimal signal:noise ratio. If the the degree of fluorescent labeling is too low, the signal will be weak. However, if the degree of fluorescent labeling is too high, the signal will also be weak due to the inactivation of the detection reagent or quenching of the signal caused by a phenomenon known as Förster resonance energy transfer (FRET).
Another concern unique with fluorescent applications is fluorescent properties of the matrix in which the proteins are immobilized on. As with fluorescence microscopy, care should be taken in choosing both the support for the proteins. Both nitrocellulose and PVDF membranes and acrylamide gels have fluorescent properties which can create a significant amount of background noise that reduces signal:noise ratios in fluorescent blotting applictions.
Data Imaging for Fluorescence
Recording and documenting the results of fluorescence-based Western blotting requires special instrumentation, namely some sort of fluorescence imager. Several manufacturers offer fluorescence imagers, most of which use either filter-based and laser-based technologies to deliver the appropriate excitation wavelength and then record the emission light output. Captured images are saved digitally (see figures on this page).
The number of commercially available fluorescent dyes having different excitation and emission spectra continues to increase. Fluors with non-overlapping spectra enable multiplex analysis, whereby two or three different targets can be detected and independently distinguished in the same lane and blot. However, the imager used must be equipped with the appropriate filters or lasers for the fluors used.
Certain instruments are specialized for detection of infrared and near-infrared fluors, while others provide for analysis of only one or two particular fluors in the visible range. Increasingly, new instruments are being offered with capabilities for detection of nearly any combination of excitation and emission wavelengths.
For additional information about instrumentation, see the links to related product and informational pages in the sidebar.
- Ex/Em Spectra: DyLight Fluor Absorption and Emission Spectra
Molecular Weight Markers for Fluorescent Detection
For detection of any Western blot, it is desirable to use prestained molecular weight markers that are transferred to the membrane along with the protein sample. The appearance of the molecular weight markers on the membrane allows estimation of molecular weights for any protein bands that are detected as well as effective separation of the proteins of interest in the gel prior to the transfer step. When chemiluminescent detection is used for Western blotting, protein bands are detected on film or with digital imaging equipment. Unless modified, molecular weight markers do not show up on during fluorescent imaging since they do not produce any signal. Therefore the use of fluorescently tagged molecular weight markers is advantageous.
Although the dyes used to make prestained molecular weight markers have some fluorescent properties, protein molecular weight markers that are labeled with fluorophores provide better signal:noise ratios. The Thermo Scientific PageRuler Prestained NIR Protein Ladder is a mixture of 10 proteins (11 to 250KDa) that are blue-stained and fluor-labeled for near-IR fluorescent visualization and protein sizing following electrophoresis. The protein MW markers in this ladder resolve into as sharp bands when analyzed by SDS-PAGE and are labeled with a fluorescent dye for visualization with instruments equipped for detection of near-infrared (NIR) fluorescence. These include Typhoon* Imagers and the LI-COR Odyssey* Infrared Imaging System.
For Research Use Only. Not for use in diagnostic procedures.