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.

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Introduction

In fluorescent western blot detection systems, signal is captured in the form of light. Transient light emission from a fluorescent molecule (fluorophore) is produced by the excitation and subsequent release of photons as the excited molecule returns back to its normal state. In contrast, chromogenic and chemiluminescent western detection systems produce signals that are products of enzyme-substrate reactions. Chromogenic enzyme-substrate reactions produce colored products that precipitate onto the membrane, while chemiluminescent detection systems generate enzymatic reactions that produce energy released in the form of light. Fluorescent applications can be more quantitative than enzyme systems. Similar to enzyme reactions, fluorescent reagents must be optimized with respect to the signal-to-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). As with fluorescence microscopy, care must be taken to select support or matrix materials that are validated for fluorescence. Typical blotting membranes such as nitrocellulose and polyvinylidene difluoride (PVDF) are known to have fluorescent properties that can cause significant background when used for fluorescent western blotting. However, reagents formulated for use for fluorescent detection have excitation and emission wavelengths that are selected to avoid auto-fluorescence from common membranes. Special low-fluorescence membranes are also available for fluorescent western blots.

Fluorescent western blotting systems are growing in popularity because they offer increased time savings over chemiluminescent detection and reduced chemical waste compared to either chemiluminescent or chromogenic detection systems. Historically, the instrumentation available for fluorescent detection has not provided the sensitivity required by many researchers or was prohibitively expensive. However, with advancements in imaging technology, new fluorescent probe development, and the reduced cost of both, fluorescent detection systems are quickly replacing chromogenic and chemiluminescent detection methods in many laboratories. While the detection limits are still not as low as chemiluminescent detection, fluorescent detection has the unique advantage of allowing multiple targets to be assayed for on the same blot at the same time without the need to strip and reprobe.

Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.

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Advantages of fluorescent western blot detection

Fluorescent methods enable multiplex western blot analysis in which several 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 Invitrogen Alexa Fluor dyes are designed for a variety of multiplex fluorescent protein and cell analysis immunoassay methods, including multiplex western detection. Recently, Invitrogen Alexa Fluor Plus secondary antibodies were developed and have up to 4.2 times higher signal-to-noise in immunofluorescence imaging and up to 5.8 times higher signal-to-noise ratio in western fluorescent blotting while having lower cross-reactivity compared to leading Alexa Fluor secondary antibodies. In addition to enabling multiplexing, fluorescent western blot detection has several other advantages compared to enzyme-based chemiluminescent substrate detection:

  • 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

Simultaneous detection of GLUT4 and GAPDH

Multicolor western detection. GLUT4 and GAPDH were detected simultaneously in 3T3-L1 adipocyte lysates using mouse anti-GAPDH (red bands) and rabbit anti-GLUT4 (green bands) with Invitrogen Alexa Fluor 680 goat anti-mouse IgG antibody and Alexa Fluor 790 goat anti-rabbit IgG secondary antibodies. Single-color images were merged to visualize both proteins. 

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Data capture for fluorescence-based western blotting

Recording and documenting the results of fluorescence-based western blotting requires special instrumentation, i.e., a fluorescence imaging instrument. Several manufacturers offer fluorescence imaging instruments, most of which use either laser or LED-based illumination with a combination of filters to deliver the appropriate excitation light wavelength and then capture the appropriate emission light output. Most instruments use a specialized charge-coupled device (CCD)–based camera to capture signal, and resulting data is captured digitally.

The diversity of commercially available fluorescent dyes with different excitation and emission spectra properties continues to increase. The wide range and spectrum of Alexa Fluor and Alexa Flour Plus dyes provide 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 imaging instrument must be equipped with an appropriate set of excitation and emission filters in order to resolve the resulting fluorescent signals. Online tools such as the Fluorescence SpectraViewer are available to assist with panel selection for multiplex fluorescence detection. Certain instruments are specialized for detection of infrared and near-infrared fluorophores, while others provide analysis of multiple fluorescent dyes in the visible range. Increasingly, modern instrumentation can combine both fluorescent imaging in the visible and near-infrared/infrared range. 

Endogenous_beta

Detection of endogenous proteins with Alexa Fluorconjugated secondary antibody.  Endogenous beta-actin (A) and endogenous MEK2 (B) was detected using rabbit anti–beta-actin and rabbit anti-MEK2 primary antibody, and Invitrogen Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 790 (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 Invitrogen XCell SureLock electrophoresis system and iBlot dry blotting system.


Detection of endogenous proteins with Alexa Fluorconjugated secondary antibody. Endogenous alpha-tubulin (a), beta-actin (b), and GAPDH (c) were 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 Invitrogen Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 680 (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.

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Using loading control antibodies in fluorescent western blotting

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, and are typically referred to as loading control proteins. The choice of which of these loading control proteins to include in western blot detection may depend on whether there is a chance that 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.

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Molecular weight markers for fluorescent detection

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-to-noise ratios. The Thermo Scientific 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 molecular weight 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. 

SDS-PAGE band profile of the PageRuler Prestained NIR Protein Ladder. The PageRuler Prestained NIR Protein Ladder was resolved on a 4-20% Tris-glycine gel (SDS-PAGE) and then visualized directly with the unaided eye (left) and via near-infrared fluorescence lasers with the Typhoon™ Imager (GE Biosciences).

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Recommended reading

  1. Towbin, H., Staehelin, T., Gordon, J. (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24:145–149.
  2. Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G. (2008) Immunoblotting and immunodetection. Curr Protoc Mol Biol. Chapter 10:10.
  3. Eaton, S. L., et al. (2013) Total protein analysis as a reliable loading control for quantitative fluorescent western blotting. PLoS One 8:72457.