Fluorescent Western Blotting

Fluorescent western blot reagents are growing in popularity because they offer time savings and reduced chemical waste compared to chemiluminescent or chromogenic detection systems. Fluorescent western blot techniques have the added bonus of being able to use multiplex detection, where multiple proteins can be detected at the same time.

Historically, the instrumentation available for fluorescent detection was not able to offer the sensitivity required by many researchers or was prohibitively expensive. However, with advancements in imaging technology, new fluorescent probes, and reduced costs, fluorescent detection systems are quickly replacing chromogenic and chemiluminescent detection methods in many laboratories.

Typical western blot detection methods involve enzyme-substrate systems that produce a colored product or emit light as a result of a chemical reaction. By contrast, fluorescent blotting methods involve detection of light emitted transiently by a fluorescent molecule (fluorophore) after it has absorbed light (excitation) and then releases photons (emission) as it returns to its normal state. This difference in signal generation allows optimized fluorescent applications to be more quantitative than enzyme systems.

Similar to enzyme reactions, fluorescent reagents must be optimized with respect to the signal:noise ratio. If 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).

Fluorescent properties of electrophoresis gel and blotting membrane materials present special concerns with fluorescent blotting applications. As with fluorescence microscopy, care must be taken to select support or matrix materials that are validated for fluorescence. Typical polyacrylamide electrophoresis gels and blotting membranes (both nitrocellulose and PVDF) are known to have fluorescent properties which can cause significant background for fluorescent western blotting.

Fluorescent methods enable multiplex western blot analysis in which multiple proteins can be simultaneously detected and differentiated on the same blot. For example, two or three different proteins can be detected via secondary antibodies conjugated to fluorescent dyes that fluoresce at different wavelengths. Secondary antibody conjugates with Alexa Fluor™ dyes are designed for a variety of multiplex fluorescent protein and cell analysis immunoassay methods, including multiplex western detection.

In addition to enabling multiplexing, fluorescent western blot detection has several other or related advantages compared to enzyme-based chemiluminescent substrate detection, despite not having quite the same level of sensitivity:

• The ability to assay multiple targets on the same blot, at the same time
• No need to strip and reprobe the blot when looking at multiple targets 
• No need to worry about substrate incubation times or film exposures
• Saves time and conserves samples
• Provides reliable quantitative data

Recording and documenting the results of fluorescence-based western blotting requires special instrumentation, i.e., some sort of fluorescence imager. Several manufacturers offer fluorescence imagers, most of which use either filter-based or 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. The wide range and spectrum of Alexa Fluor™ dyes provides options for many filter and multiplex detection needs. Fluorophores 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 fluorophores, while others provide analysis of only one or two particular fluorescent dyes in the visible range. Increasingly, new instruments are being offered with capabilities for detection of nearly any combination of excitation and emission wavelengths. Alexa Fluor™ dyes can be used with various instruments, including LI-COR™ Odyssey™ Imaging Systems (LI-COR, Inc.).

Figure 2:  Endogenous beta-actin (A) and endogenous MEK2 (B) was detected using rabbit anti–beta-actin and rabbit anti-MEK2 (Cat. No. 700829) primary antibody, and goat anti-rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor™ 790 Conjugate (Cat. No. A27041) (0.4 µg/mL 1:2,500 dilution). No primary control to assess background (C) and loading control (LC) mouse anti-Tubulin (Cat. No. 322500) is shown. Western blot analysis was performed on whole cell extracts (20 µg lysate) of HeLa human cervical carcinoma cells (lanes 1–5) using the XCell SureLock™ electrophoresis system and iBlot™ Dry Blotting System.

Figure 3: Endogenous alpha-tubulin (a), beta-actin (b), and GAPDH (c) was detected at 52 kDa, 46 kDa, and 37 kDa respectively using mouse anti–alpha-tubulin (Cat. No. 322500), beta-actin (Cat. No. AM4302), and mouse anti-GAPDH (Cat. No. 398600) primary antibody and goat anti-mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor™ 680 Conjugate (Cat. No. A28183) (1 µg/mL 1:1,000 dilution). Western blot analysis was performed on whole cell extracts (20 µg lysate) of HeLa human cervical carcinoma cells (lane 1–5) using the XCell SureLock™ electrophoresis system and iBlot™ Dry Blotting System.

Because the signal output from fluorescent western blotting is proportional to the amount of protein present, it is possible to make quantitative measurements from western blot experiments. This aspect of western blotting can be useful when looking at treatments that cause changes in expression levels of proteins. However, to verify that any change is the result of the treatment and not changes in the amount of sample loaded, a housekeeping protein, normally expressed at consistent levels, is used to normalize results.

GAPDH, beta-actin, and beta-tubulin are housekeeping proteins that are commonly used for loading normalization. The choice of which of these loading control proteins to include in western blot detection may depend upon whether there is a chance any treatment would impact that housekeeping protein. When running the experiment, a researcher usually completes a western blot with an antibody to the target of interest in one color and an antibody to the normalizing protein in a second color. The fluorescent intensities are measured and then expressed as a ratio of target intensity to normalizing protein intensity. When running these multiplexed experiments, it is important to choose secondary antibodies that do not cross-react. Pre-conjugated loading control antibodies can be used to simplify these quantitative experiments by removing the need for a secondary antibody for these highly abundant targets.

For detection of any western blot, it is desirable to use prestained molecular weight markers (also called protein ladders) 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 and verification of the effective separation of the proteins of interest in the gel prior to the transfer step. Typical protein molecular weight markers will not be visible (detected) during fluorescent imaging because they do not produce any fluorescence signal. Fluorescently tagged molecular weight markers must be used.

Although the dyes used to make prestained molecular weight markers often have some fluorescent properties, protein molecular weight markers that are labeled with fluorophores provide better signal:noise ratios. The PageRuler™ Prestained NIR Protein Ladder is a mixture of 10 proteins (11 to 250 kDa) that are blue-stained and fluorophore-labeled for near-infrared fluorescent visualization and protein sizing following electrophoresis. The protein MW markers in this ladder resolve 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 (GE Healthcare) and the LI-COR™ Odyssey™ Infrared Imaging System (LI-COR, Inc.).