With billions of neurons connected by trillions of synapses, the brain is by far the most intricate and complex organ. A complete map of the brain’s connections will be essential to understanding its function. Some of the tools that have been invaluable towards creating this map are the colorful range of fluorescent proteins used to label individual neurons and subcellular structures. Until recently, though, only about three different fluorescent colors could be used at the same time, limiting the range of labeling options, especially in regions that were densely-populated or had thousands of intertwined neuronal projections. In these cases, the low number of different colors made it difficult to distinguish adjacent cells. Fortunately, a fluorescence-based strategy called “Brainbow” in mice allows for a much larger spectral range. Brainbow uses several cassettes of multi-colored fluorescent protein transgenes in combination with Cre-lox genetic recombination to randomly express one fluorescent protein gene per cassette. The fluorescent colors thus mix to make an individual cell fluoresce with one of up to 90 possible colors, allowing for easy differentiation of adjacent neurons. On its own, this technique creates informative images, and its signal intensity can be improved even further with immuno-labeling. One challenge to this immune strategy, though, has been that many of the different colors of fluorescent protein, such as those derived from the original jellyfish green fluorescent protein, have very similar sequences and are thus indistinguishable from each other as antigens. Therefore, differently-colored fluorescent proteins would have a strong advantage if they were also antigenically distinct from each other.
Fluorescent proteins with minimal sequence homology improve both signal intensity and experimental efficiency
To identify fluorescent proteins that were antigenically distinct, the amino acid sequences of fluorescent proteins from various marine animals were screened. A group of five differently-colored proteins that had minimal sequence similarity were chosen for affinity and size exclusion purification to reduce cross-reaction and avoid increased background signal. These proteins were then used as antigens for immunization in five different animal host species. Because the resulting fluorescent protein antibodies do not interfere with the reactivity of one another, they can all be used in the same incubation. Therefore, experiments such as those visualizing complex neuronal processes not only have enhanced fluorescence signal intensity, but they have shorter experimental times, as well.
- Chromatin immunoprecipitation (ChIP) assays
- Western blots
- Flow cytometry
- Affinity purification
- Enhanced fluorescence signal-to-background ratio
- Faster experiments from simultaneous use of multiple antibodies