fluorescent proteins by james dicarlo and ingrid spielman august 12, 2008 aequorea victoria gfp...
TRANSCRIPT
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