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

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

3. p-hydroxybenzylidene-imidazolidone Stokes Shift To fall back to Ground Level State: Vibrational relaxation Rotational energy Emission 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 Conjugated Double Bonds

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

7. Our FPs ColorWavelength violet380–450 nm blue450–495 nm green495–570 nm yellow570–590 nm orange590–620 nm red620–750 nm GFP 395 /509 nm mCherry 587/610 Venus YFP502/532 Sapphire 399/508

9. Immunofluorescence 1)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

10. 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

11. 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