Fluorescence is a direct imaging method that uses fluorescent dyes – also known as fluorophores or probes – that are attached to a secondary antibody. While approximately 80% of researchers continue to use enzyme-based chemiluminescence for their Western blots, fluorescent imaging is quickly gaining ground as a preferable method due to improvements in imaging and fluorophore technologies and more abundantly available antibodies. Fluorescence offers several advantages over traditional chemiluminescence, including: 1) the ability to multiplex target proteins without necessarily the need to strip-and-reprobe, 2) enhanced quantitative detection, and 3) the ability to archive and reimage blots due to the stability of fluorescent molecules. Most Western protocols can be easily adapted from chemiluminescent or colormetric methods, but some optimization is required. Below are some techniques to avoid common pitfalls when using fluorescent detection.
Multiplexing – Detect One Protein at a Time
Most researchers limit multiplexing to two proteins, although it is possible to detect three proteins (or more) with today’s technology. The more proteins combined on a blot, the more complex optimization becomes. Finessing detection of each target protein before simultaneously detecting multiple targets is therefore recommended. Single target detection helps determine the banding pattern of each antibody prior to multiplex analyses. It’s advisable to detect the strongest target protein with the blue channel, the middle target with the green channel, and weaker proteins with the red channel. (Membranes have higher inherent fluorescence with short wavelength excitation (blue).) Also, always use primary antibodies from different species – e.g. mouse to goat – as antibodies produced from two closely related species – e.g. mouse to rat – may cross-react.
Avoiding Cross-Channel Fluorescence
A common problem in multiplex studies – especially in post-translational modification studies where the proteins are similar in weight and migrate closely together – is cross-channel fluorescence or bleed-through. Bleed-through is when you image for blue but pick up bands on, for instance, the Cy3 (green) channel. To avoid cross-talk, researchers should verify that secondary antibodies are bound to fluorophores with non-overlapping emission spectra. It’s also worth double-checking emission filters before acquiring images as filters are designed to cover specific band passes.
Low Overall Signal – Increase Antibody Concentrations for Best Results
One challenge in adapting a chemiluminescence protocol for fluorescence is revising antibody concentrations. Typically, primary antibody concentrations should increase two- to five-fold over standard chemiluminescent protocols for proper signal. However, an extremely high antibody concentration can also increase background noise. (Ideally, you want low background noise, which yields a higher signal-to-background ratio.) High antibody concentrations can adversely lead to nonspecific binding as well. Optimizing antibody concentrations is then critical for best results. An easy way to experiment with concentrations is to incubate the membrane in a variety of dilutions for each antibody. Secondary antibodies may also require optimization – a 1:5,000 dilution is a good starting point.
Minimizing Autofluorescence
With chemiluminescence, the proportion of light emitted correlates to the local enzyme-substrate concentration. No direct light source is required for exposure -- more enzyme yields more light in what is a time-limited reaction. As a result, the target signal never has to compete with autofluorescence across the membrane. (This is one reason chemiluminescence is reportedly more sensitive than fluorescence when working with low protein levels.) An easy solution to this problem is substituting low fluorescence PVDF membranes for standard PVDF membranes when using fluorescent detection. These membranes increase signal-to-background ratios per channel for transferred proteins, producing bright clean bands. To additionally minimize fluorescent artifacts, avoid using inks to mark membranes, don’t handle while wearing powdered nitrile gloves, and make sure to be gentle when handling membranes. While there’s no risk to photobleaching fluorescently labeled antibodies in working daylight conditions, all fluorescently labeled antibody stocks should be stored in the dark.
Your Reward: Clear Images and Strong Results
In conclusion, it’s worth noting that all fluorescent imaging systems are digital. One of the joys in working with fluorescent imaging is the astounding publication-ready images researchers can easily capture. While the entry price for a digital imager might be prohibitively high for many labs – digital imagers start at around $30K and run upwards of $100K – the price per reaction drops significantly thereafter in comparison to chemiluminescence. As more researchers multiplex and demand better linearity, fluorescent imaging for Western blotting is likely to become an industry standard in the years to come.
The image at the top of the page was provided by Bio-Rad's Western Blotting group.