Improvements in imaging and fluorescent probes provide scientists with enhanced tools to maximize the utility of western blot technology. Fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to evaluate multiple protein targets on a single blot, which is known as multiplexing. Multiplexing helps make research more efficient and productive. For example, one can visualize a protein of interest simultaneously with a loading control protein (Figure 1), differentiate proteins of similar molecular weights (Figure 2), and evaluate complex biological pathways (Figure 3). In multiplexing terminology, probing for 2 proteins of interest is known as a 2-plex experiment, probing for 3 proteins of interest is known as a 3-plex experiment, etc. With the Invitrogen™ iBright™ FL1000 Imaging System, one can perform up to a 4-plex fluorescent western blot with the appropriate experimental setup (Figure 4). This guide shares best practices and tips for success in multiplex fluorescent western blotting.

Important Considerations Before You Get Started

Before setting up your multiplexing experiment, consider these points to help minimize background fluorescence:

• To eliminate a major source of background fluorescence, use transfer membranes with low autofluorescence, such as Thermo Scientific™ nitrocellulose membranes (Cat. No. 88018 or LC2001) or low-fluorescence polyvinylidene difluoride (PVDF) membranes (Cat. No. 22860).
• Only handle membranes with gloved hands and clean blunt forceps to limit contamination and scratches on the membrane, which can contribute to background fluorescence and other fluorescent artifacts.
• Use high-quality, filtered buffers (e.g., Thermo Scientific™ Blocker™ FL Fluorescent Blocking Buffer (Cat. No. 37565)). Particles and contaminants in blocking and wash buffers can settle on membranes and create fluorescent artifacts. In addition, limit the use of detergents during blocking steps, as common detergents can autofluoresce, possibly increasing nonspecific background.
• Sample buffers may contain components that fluoresce (such as bromophenol blue), which can contribute to increased background. We recommend using fluorescence-compatible sample buffers (e.g., Invitrogen™ Fluorescent Compatible Sample Buffer, Cat. No. LC2570).
• If molecular weight markers contain proteins labeled with fluorophores, or that are stained with compounds that may fluoresce at certain wavelengths, decrease the volume of markers loaded onto the gel. Overloading may obscure the signal in adjacent lanes. Consider the Invitrogen™ iBright™ Prestained Protein Ladder (Cat. No. LC5615) for your multiplexing setup. Typically, 2–4 µL of the iBright Prestained Protein Ladder is sufficient for visualization and fluorescence detection.

Figure 1: Simultaneous Detection of Protein of Interest and Loading Control

Figure 1. Simultaneous detection of protein of interest and loading control protein. The signal of each protein is captured in a different fluorescence channel, which enables the detection of two proteins on a single blot without stripping and reprobing. Composite image shown, overlaying the signals from each probe.

Figure 2: Detection of Targets of Similar Molecular Weights

Figure 2. Detection of targets of similar molecular weights. Fluorescent multiplexing allows for 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.

Figure 3: Study Complex Biological Signaling Pathways

Figure 3. Study complex biological signaling pathways. Western blot analysis of the phosphorylation of EGFR and its downstream target, MEK1, after EGF treatment of A431 EGFR knockout (KO) cells.

Figure 4: A 4-Plex Western Blot

Figure 4. A 4-plex western blot imaged on the iBright FL1000 system.

Selection of Antibodies

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.

? Tip: Look for antibodies for which the supplier provides target-specific verification and validation to ensure the antibodies bind to the right target and to also save time validating. Learn more about the 2-step validation* testing methodology for Invitrogen™ antibodies at thermofisher.com/antibodyvalidation

* The use or any variation of the word "validation" refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.

Filter Sets and Fluorophore Selection

In a multiplex western blot, ideally each target protein is captured independently in separate images under conditions that eliminate any cross-talk 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 (see Table 1 for the preinstalled filter sets of the iBright FL1000 Imaging System). Use a tool like the Fluorescence SpectraViewer (thermofisher.com/spectraviewer) to determine excitation and emission spectral overlap among the fluorophores available to you, 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). An example of two fluorophores with a high degree of excitation and emission spectral overlap is shown in Figure 5. An example of a combination of fluorophores with minimal excitation spectral overlap is shown in Figure 6. An example of a combination of fluorophores with minimal emission spectral overlap is shown in Figure 7.

Table 1: iBright FL1000 Imaging System Filter Sets

Note: Avoid using the channels EX3/EM3 and EX4/EM4 together in a multiplex experiment, because of the high degree of spectral overlap of fluorophores that would be captured in these channels.

? Tip: When imaging with the iBright FL1000 Imaging System, use the brighter, low-wavelength fluorophores for low-abundance targets (e.g., Invitrogen™ Alexa Fluor™ 546 and Alexa Fluor Plus 647 probes) and, conversely, higher-wavelength fluorophores (e.g., Alexa Fluor Plus 680 and Alexa Fluor Plus 800 probes) for high-abundance targets.

Figure 5: Example of Significant Spectral Overlap

Figure 5. Example of significant spectral overlap. In this example generated on our online Fluorescence SpectraViewer, portions of the excitation and emission spectra of both Alexa Fluor Plus 647 and Alexa Fluor 680 fluorophores are within the ranges of the excitation and emission filters, so both fluorophores would be excited and their emissions would reach the camera's detector under these conditions, making it difficult to distinguish the source of the detected fluorescence.

Figure 6: Example of Multiplex Experiment with Distinct Excitation Spectra

Figure 6. Example of multiplex experiment with carefully chosen fluorophores with distinct excitation spectra. In this example generated on the Fluorescence SpectraViewer, the excitation spectra (dashed lines) of Alexa Fluor Plus 488 and Alexa Fluor 546 fluorophores have minimal overlap within the range of the excitation filter. Despite both fluorophores having part of their emission spectra (solid lines) within the range of the emission filter, Alexa Fluor 546 would not be excited by the excitation filter that has been selected for Alexa Fluor Plus 488, so no fluorescence from Alexa Fluor 546 would be present to go through the emission filter.

Figure 7: Example of Multiplex Experiment with Distinct Emission Spectra

Figure 7. Example of multiplex experiment with carefully chosen fluorophores with distinct emission spectra. In this example generated on the Fluorescence SpectraViewer, the emission spectra (solid lines) of Alexa Fluor Plus 680 and Alexa Fluor 790 have no overlap within the ranges of the two emission filters. Despite both fluorophores having part of their excitation spectra (dashed lines) within the range of excitation filter 1, any Alexa Fluor 790 fluorescence generated by that excitation range is not within the wavelengths allowed to pass through emission filter 1, so no fluorescence from Alexa Fluor 790 would reach the camera detector in that channel.

General Procedure for Multiplex Fluorescent Western Blotting

Troubleshooting