Fluorescent Western Blotting

Fluorescent western blotting is growing in popularity because it allows the ability to perform 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. With the introduction of advanced digital imaging instruments like the Invitrogen iBright FL1500 Imaging System, and improvements in fluorescent conjugate technologies, scientists now have the necessary tools to take advantage of the range of fluorescent dyes and antibodies for western blot detection. These advancements provide access to fluorescence detection with reduced cost and improved sensitivity.

Explore: Reagents for fluorescent western blotting  Fluorescent Western Blot ProtocolExplore: Fluorescent imaging systems

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. Overall, the western blotting procedure is similar between chemiluminescent and fluorescent detection methods, with each method offering specific benefits.

Similar to enzymatic 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 can 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).

Fluorescence vs chemiluminescence western blot detection

While the detection limits are not as low as chemiluminescent detection, fluorescent detection has the unique advantage of allowing multiple targets to be assayed on the same blot at the same time without the need to strip and reprobe. Multiplexing helps make research more efficient and productive. For example, one can visualize a protein of interest simultaneously with a loading control protein, or differentiate proteins of similar molecular weights, or evaluate complex biological pathways. With the iBright FL1500 Imaging System, one can perform up to a 4-plex fluorescent western blots with the appropriate experimental setup.

In addition to enabling multiplexing, fluorescent western blot detection has several other advantages compared to enzyme-based chemiluminescent substrate detection. Fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to conserve sample due to multiplexing.

Advantages of fluorescent detection

• Greater quantifiable linear range for quantitative western blotting techniques
• No need to strip and reprobe the blot when looking at multiple targets
• Stable signals
• Ability to save sample with multiplexing

Detection of targets of similar molecular weights. Fluorescent multiplexing allows clear distinction of multiple targets on the same blot, even when they are of similar molecular weights. A composite image is shown along with images showing the single-color signals of individual proteins. Visualizing the individual signals can sometimes enable assessment of details that may be harder to see in a composite.

When getting started with fluorescent western blotting, some reagents and steps will need to be optimized to help ensure background fluorescence does not interfere with detection of the protein of interest. Here are some tips for getting started:

• Sample buffers containing bromophenol blue will fluoresce and can contribute to increased background. While the dye front may be run off the gel prior to transfer or cut from the membrane after transfer to avoid background fluorescence signal, it is ideal to use fluorescence-compatible sample buffers without bromophenol blue, such as Fluorescent Compatible Sample Buffer.

• Decrease the amount of molecular weight markers loaded onto the gel. Standard prestained molecular weight markers can be used, but the loading amount will need to be optimized if the marker contains fluorescent bands since overloading can increase background fluorescence and signal bleed-through to adjacent lanes. The iBright Prestained Protein Ladder (Cat. No. LC5615) provides prestained proteins as well as fluorescent bands for detection. Typically, 2–4 µL of the iBright Prestained Protein Ladder is sufficient for visualization and fluorescence detection.
• To eliminate a major source of background fluorescence, use transfer membranes with low autofluorescence, including nitrocellulose and specialty low-fluorescence PVDF membranes such as Nitrocellulose Membrane (Cat. No. 88018) and Low-Fluorescence PVDF Transfer Membrane (Cat. No. 22860).

• Use only high-quality filtered buffers, such as Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565). Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. In addition, limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and increase nonspecific background.
• Only handle membranes with gloved hands and clean blunt forceps to limit contamination and scratches on the membranes, which can contribute to background fluorescence and artifacts. Avoid using pens on membranes, as many inks fluoresce.
• Secondary antibody concentrations are typically higher in fluorescence applications. Optimization is required to achieve the best signal-to-noise ratio, but the recommended concentration range, regardless of fluorescent conjugate, is typically between 0.4 and 0.1 µg/mL (1:5,000–1:20,000) for imaging on CCD imaging systems. Alexa Fluor Plus secondary antibodies were designed to provide high signal-to-noise ratios and lower cross reactivity, reducing the time needed for optimization.

Explore:Fluorescence friendly reagents

Selection of antibodies for multiplex western blotting

The selection of appropriate primary antibodies and fluorescently labeled secondary antibodies is critical when designing a fluorescent multiplex western blot experiment. Here are some guidelines to consider:

• Select antibodies designated specifically for western blotting or that list western blotting as an application. Verify the detection of each protein target individually before multiplexing with other targets. This will allow determination of the banding pattern of each antibody prior to a multiplex experiment.
• Use primary antibodies from different host species for each target being probed. Ideally, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep. This will aid in the selection of appropriate secondary antibodies to minimize potential antibody cross-reactivity, which can lead to confusing results.
• If your protein is epitope tagged (e.g., 6xHis, HA), you may be able to take advantage of a fluorophore conjugated primary antibody specific to the epitope tag. A primary antibody directly conjugated to a fluorophore does not require the use of a fluorophore-conjugated secondary antibody. This can minimize the number of secondary antibodies needed in a multiplexing experiment, but careful consideration of primary antibody host species is still required to prevent secondary antibody cross-reactivity with the other primary antibodies used in the experiment. Fluorescent labeling kits are also available if particular direct conjugates are not commercially available.
• If differentiation of the primary antibody host species is difficult, consider antibodies that are of a single specific antibody class (IgM, IgG, etc.) or isotype (IgG1, IgG2a, etc.), and a relevant secondary antibody that recognizes only that specific host class or isotype. If you are unsure of the specificity of your antibodies, contact your antibody supplier for more information.
• Additionally, consider using secondary antibodies that are highly cross-adsorbed. Cross-adsorption is a purification process that helps eliminate nonspecific antibodies in an antibody mixture, such as antibodies of specific classes, isotypes, or host species. If you are unsure of the potential cross-reactivity of your secondary antibodies, contact your antibody supplier for more information.

Explore:Antibodies

In a multiplex western blot, ideally each target protein is captured independently in separate images under conditions that eliminate any crosstalk between the fluorescent probes. Therefore, it is essential to know the configuration of the western blot imaging instrument before you begin, most importantly the available excitation and emission filter sets. Many imaging systems use a combination of excitation and emission filter sets that can be chosen to allow a narrow range of light wavelengths to pass through to excite the fluorophore and for the specific fluorophore emission to enter the camera’s detector. Instead of filter sets, some instruments may use independent narrow-spectrum light sources for excitation. The specific combination of excitation and emission conditions used is often referred to as a “channel” or “layer” and determines what fluorescent probes can be imaged separately. Depending on the instrument, the available filters may come preinstalled or require installation.

Use a tool like the Fluorescence SpectraViewer to determine excitation and emission spectral overlap among the fluorophores available, in the context of the specific imaging instrument’s equipped excitation and emission filters. Ideally, the fluorophores used in a multiplex experiment have distinct regions of either excitation or emission spectra that are compatible with the imaging system’s filters. Note, only a region of either the excitation or the emission spectrum needs to be distinct (not both).

Examples of multiplex fluorophore combinations that can be used with the iBright FL1500 Imaging System

Secondary antibody conjugates with Invitrogen Alexa Fluor Plus dyes are designed for a variety of multiplex fluorescent protein immunoassay methods, including multiplex western detection. Alexa Fluor Plus secondary antibodies 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.

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, the protein target signals must be normalized.

Normalization has been traditionally performed using a housekeeping protein, normally expressed at consistent levels 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 depends on whether there is a chance that any experimental treatment impacts the expression of the housekeeping protein. When detecting housekeeping proteins, a different conjugate or color is used from the target protein detection. 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.

Housekeeping proteins can often be affected by experimental conditions and can often have oversaturated western blotting signals due to high abundance. In these cases, total protein normalization is a better strategy for quantitative western blotting. Total protein analysis is independent of the shortfalls of loading control proteins, where the total protein loaded is used to normalize the target signals. Total protein loads can be determined using several different protein membrane stains or labeling reagents, such as No-Stain Protein Labeling reagent. It is important when normalizing to normalize to total protein loads on the membrane to account for variable transfer efficiencies.

Continue reading:
Normalization using No-stain Protein Labeling Reagent
Normalization using loading control proteins

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. The dyes used to make prestained molecular weight markers often have some fluorescent properties, however their signals can potentially overwhelm the fluorescence of the target proteins. Using protein ladders specifically designed for fluorescent western blotting can help balance the fluorescent signals. In addition, protein molecular weight markers that are labeled with fluorophores can provide better signal-to-noise ratios.

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.

• 5-steps to Multiplexed Fluorescent Western Blotting
• Webinar: Light up your western blots- fluorescent western blotting tips, tricks & more
• Fluorescent Western Blotting – an introduction for new users
• Application Note: Fluorescent Western Blotting – a guide to multiplexing
• Application Note: Normalization in Western Blotting to achieve relative quantitation
• Handbook: Protein Gel Electrophoresis Technical Handbook
• Handbook: Western Blotting Technical Handbook
• Handbook: Protein Research