Lesson 13
Fluorescent Protein Assays
Fluorescent probes (fluorophores) are often used to study protein binding and localization. Fluorophores are compounds that absorb energy at a specific wavelength and emit energy at a higher wavelength to return to their ground (original) energy state (this process is called fluorescence). There are many techniques which use fluorescence, and only a few will be introduced here.
Fluorescent Tags
Fluorescent tags can be recombinantly added to proteins when designing their DNA. Tags can be chemically bound to a protein or molecule by specific chemical reactions. Fluorescent tags can also be covalently bound to protein antibodies. These antibodies will bind specifically to unique proteins and thus specifically fluorescently label the protein of interest. Fluorescence experimental assays can be either in vivo (in whole cells) or in vitro (in a test tube using recombinant proteins).
Whole Cell Assays
Fluorescently tagged proteins can be imaged in whole cells. One way to do this is to individually inspect each cell using a high power fluorescence microscope. However, this can take a long time and not give a large amount of data. Another way is to use flow cytometry where cells are individually imaged and analyzed by lasers. Then, computer programs are used to quantitatively analyze the data. Watch this video on flow cytometry.
A newer technique in whole cells is single molecule fluorescence using super-resolution microscopy. Using this technique, single molecules can be traced over time in the cell. Read about some of the interesting research being conducted in the Gahlmann Lab at UVA using cell imaging.
FRET
Förester resonance energy transfer (FRET) can be used to determine if two molecules are within a certain distance of one another, which can indicate a molecular interaction. Fluorophores are compounds that absorb energy at a specific wavelength and emit energy at a higher wavelength to return to their ground (original) energy state. Different fluorophores accept and emit light at different wavelengths. Two fluorophores are considered a FRET pair if the wavelength that one emits energy is within the same wavelength range that the other accepts energy (see this article for an energy diagram of what this looks like). When one fluorophore (called the donor) is excited by a light source the energy is transferred to the second fluorophore (called the acceptor) which emits light at a different wavelength (emitting fluorescence). This process can only occur when the two FRET pair fluorophores are within a certain distance of one another (the actual distance depends on the specific pair and is called the Förester radius). If the FRET pair are too far apart, the energy will not be transferred and you will not observe acceptor fluorescence. FRET can be used both with in vivo (whole cell) and in vitro (test tube) experiments.
Assignment: Will will have a journal club this week, looking at a paper (Kitamura et al., 2018) presenting a FRET based assay that was developed for LspA (note that this paper uses Lsp instead of LspA). This assay is a unique usage of FRET. Normally, FRET is used to identify interactions when the FRET pairs are close together and FRET emission occurs. In this paper, one molecule of the FRET pair is a quencher (meaning it uses up or absorbs all of the energy) and so there is no fluorescence when the FRET pair is close together. When the quencher moves away from the fluorophore, the fluorophore is able to fluoresce. Thus, in this assay, fluorescence occurs when the pair is far apart (signaling Lsp cleavage in this case).
Each person will present one figure to the group. Remember to think about if the research question is sound, if the experimental design makes sense, and if the data presented supports the author's conclusions.