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Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008

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This article explores the fascinating world of fluorescent proteins, focusing on GFP (Green Fluorescent Protein) and its derivatives like mCherry and Venus YFP. It highlights the structural features such as conjugated double bonds and quantum efficiency that contribute to their fluorescence. The mechanisms for achieving fluorescence via specific amino acid sequences like Ser-Tyr-Gly are also discussed, along with the applications of these proteins in immunofluorescence and FRET (Fluorescence Resonance Energy Transfer). The pros and cons of different methods are reviewed, providing insights into their usage in cellular studies.

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Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008
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  1. Fluorescent ProteinsBy James Dicarlo and Ingrid SpielmanAugust 12, 2008 GFP chromophore Aequorea victoria

  2. Tons of different Fluorescent Proteins Shape Discosoma is a coral

  3. Conjugated Double Bonds p-hydroxybenzylidene-imidazolidone Stokes Shift • Conjugated double bonds: • Ring Structure • Pi bonds • The more the merrier: lower energy photons required (visible light) • Quantum efficiency increases as number of pi bonds increase • To fall back to Ground Level State: • Vibrational relaxation • Rotational energy • Emission

  4. How can a Ser-Tyr-Gly sequence fluoresce?

  5. IDEAL FPs? • Sufficient signaling: Brightness measured by quantum yield; the ratio of photons absorbed to photons emitted • High photostability: does not bleach out easily • Minimal cross-talk between excitation and emission • Fold in vivo at certain pH and temperature levels • Should not oligomerize: can engineer into monomers via A206K mutation • Not toxic

  6. Our FPs • GFP 395 /509 nm • mCherry 587/610 • Venus YFP 502/532 • Sapphire 399/508

  7. A Rabbit or Goat antibody is created to • the antigen of interest • 2) A secondary antibody is based on • the primary antibody. • 3) The Primary antibody is bound • to the antigen of interest and then • the secondary (dye coupled) antibody • is used to locate the primary antibody. • Pros-This method is useful for locating different cellular bodies but use the same secondary dye coupled antibody. • Cons- This method interferes with cell function while fusion GFP constructs allow for viewing of intact cell/macromolecule • http://www.bio.davidson.edu/COURSES/genomics/method/IMF.html Immunofluorescence

  8. FRET • Fluorescence Resonance Energy Transfer • Emission wavelength of one fluorescent protein (or other chromophore) serves as Absorption wavelength for other Protein http://en.wikipedia.org/wiki/Fluorescence_resonance_energy_transfer

  9. FACS • Fluorescence Activated Cell Sorting • Laser is passed through flowing liquid to excite chromophores of a Specific wavelength. • Can determine density and composition of mixture • Multiple lasers can be used to separated different populations of cells http://www.invitrogen.com/site/us/en/home/support/Tutorials.html