Fluorescent Proteins By James Dicarlo and Ingrid Spielman

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

Tons of different Fluorescent Proteins Shape Discosoma is a coral

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

How can a Ser-Tyr-Gly sequence fluoresce?

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 p. H and temperature levels • Should not oligomerize: can engineer into monomers via A 206 K mutation • Not toxic

Our FPs • GFP • m. Cherry • Venus YFP • Sapphire 395 /509 nm 587/610 502/532 399/508 Color Wavelength violet 380– 450 nm blue 450– 495 nm green 495– 570 nm yellow 570– 590 nm orange 590– 620 nm red 620– 750 nm

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

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

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