Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP...
-
Upload
homer-cook -
Category
Documents
-
view
215 -
download
0
Transcript of Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP...
![Page 1: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/1.jpg)
Fluorescent ProteinsBy James Dicarlo and Ingrid Spielman
August 12, 2008
Aequorea victoria
GFP chromophore
![Page 2: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/2.jpg)
Tons of different Fluorescent Proteins
Shape Discosoma is a coral
![Page 3: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/3.jpg)
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
![Page 4: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/4.jpg)
How can a Ser-Tyr-Gly sequence fluoresce?
![Page 5: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/5.jpg)
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
![Page 6: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/6.jpg)
![Page 7: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/7.jpg)
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
![Page 8: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/8.jpg)
![Page 9: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/9.jpg)
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
![Page 10: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/10.jpg)
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
![Page 11: Fluorescent Proteins By James Dicarlo and Ingrid Spielman August 12, 2008 Aequorea victoria GFP chromophore.](https://reader035.fdocuments.in/reader035/viewer/2022081519/56649f1f5503460f94c37b40/html5/thumbnails/11.jpg)
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