Fluorescent ProteinsBy James Dicarlo and Ingrid Spielman

August 12, 2008

Aequorea victoria

GFP chromophore

Tons of different Fluorescent Proteins

Shape Discosoma is a coral

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

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 pH and temperature levels• Should not oligomerize: can engineer into

monomers via A206K mutation• Not toxic

Our FPs

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

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

Immunofluorescence1) A Rabbit or Goat antibody is created to the antigen of interest

2) A secondary antibody is based onthe 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