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S. Semih EKIMLER
Aims
   Applications with fluorescent proteins

   Properties of fluorescent proteins

   Reasons for upgrading fluores...
Why do we use fluorescent
proteins?
 To track and quantify proteins
 To watch protein-protein interactions
 To describe...
Characteristics of
Fluorescent Proteins
 Expressed efficiently
 No phototoxicity
 Bright enough
 Sufficient photostabi...
Reasons for New FPs
   For brighter fluorescence
         improving quantum yield
         higher extinction coefficient
...
Reasons for New FPs
   To change absorbance and emission
    spectra
        less spectral overlap
        better spectra...
Reasons for New FPs
   Less sensitive to environment
        pH resistance
        ions

   Deeper tissue penetration
New Fluorescent Proteins
   The discovery of GFP from jellyfish




   Mutagenesis studies on GFP
    New fluorescent pr...
Blue Fluorescent Protein
(BFP)
 Shifts in absorbance and emission spectra
 First used in multicolour imaging and FRET


...
Cyan Fluorescent Protein
(CFP)
 Has a spectra between BFP and eGFP
 Brighter
 Displays more photostability
Cyan Fluorescent Protein
(CFP)
 A new version of CFP      Cerulean
 Brighter
 Improves the signal/noise of FRET
Yellow Fluorescent Protein
(YFP)
 The absorption amd emission spectra
  are shifted to red wavelengths
 Imaging partner ...
Yellow Fluorescent Protein
(YFP)
   Citrine and Venus

   Chloride sensitivity eliminated

   Sensitivity to pH changed...
Red Fluorescent Proteins
(RFP)
 From other marine organisms
 Discosoma        DsRed
  Heteractis crispa     HcRed
 Most...
Red Fluorescent Proteins
(RFP)
 DsRed
        needs incubation at 37ºC
        obligate tetramer
 Minimizing oligomeriza...
New Fluorescent Proteins
 New approach for monomeric red
  fluorescent proteins
 Replacing N terminus of mRFP1 with GFP
...
New Fluorescent Proteins
 All variants are brighter than mRFP1
  (except mHoneydew, mBanana,
  mTangerine)
 mOrange is t...
New Fluorescent Proteins
   Protein lifetimes and turnover rates

   First little initial fluorescence with
    excitati...
New Fluorescent Proteins
   PA-GFP      developed from GFP
                increase in fluorescence
  when illuminated at...
Summary
 We use the fluorescence proteins to see
  changes in cells
 Fluorescence proteins have common
  properties
 Im...
References
   Lippincott-Schwartz, J., et al., 2003. Development adn Use of Fluorescent Protein
    Markers in Living Cel...
New Fluorescent Proteins   S. Semih Ekimler(3)
New Fluorescent Proteins   S. Semih Ekimler(3)
New Fluorescent Proteins   S. Semih Ekimler(3)
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New Fluorescent Proteins S. Semih Ekimler(3)

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Transcript of "New Fluorescent Proteins S. Semih Ekimler(3)"

  1. 1. S. Semih EKIMLER
  2. 2. Aims  Applications with fluorescent proteins  Properties of fluorescent proteins  Reasons for upgrading fluorescent proteins  Examples of new fluorescent proteins
  3. 3. Why do we use fluorescent proteins?  To track and quantify proteins  To watch protein-protein interactions  To describe biological events and signals in a cell
  4. 4. Characteristics of Fluorescent Proteins  Expressed efficiently  No phototoxicity  Bright enough  Sufficient photostability  No oligomerization  Minimal overlap in excitation and emission profile
  5. 5. Reasons for New FPs  For brighter fluorescence improving quantum yield higher extinction coefficient quicker maturation
  6. 6. Reasons for New FPs  To change absorbance and emission spectra less spectral overlap better spectral separation  Longer fluorescence lifetime  Less photobleach
  7. 7. Reasons for New FPs  Less sensitive to environment pH resistance ions  Deeper tissue penetration
  8. 8. New Fluorescent Proteins  The discovery of GFP from jellyfish  Mutagenesis studies on GFP New fluorescent proteins
  9. 9. Blue Fluorescent Protein (BFP)  Shifts in absorbance and emission spectra  First used in multicolour imaging and FRET BUT,  Dim  Photobleach easily
  10. 10. Cyan Fluorescent Protein (CFP)  Has a spectra between BFP and eGFP  Brighter  Displays more photostability
  11. 11. Cyan Fluorescent Protein (CFP)  A new version of CFP Cerulean  Brighter  Improves the signal/noise of FRET
  12. 12. Yellow Fluorescent Protein (YFP)  The absorption amd emission spectra are shifted to red wavelengths  Imaging partner of CFP (FRET)
  13. 13. Yellow Fluorescent Protein (YFP)  Citrine and Venus  Chloride sensitivity eliminated  Sensitivity to pH changed  Photobleaching improved
  14. 14. Red Fluorescent Proteins (RFP)  From other marine organisms  Discosoma DsRed Heteractis crispa HcRed  Most suitable red markers
  15. 15. Red Fluorescent Proteins (RFP)  DsRed needs incubation at 37ºC obligate tetramer  Minimizing oligomerization red fluorescent tandem dimers  Mrfp1 completely monomeric matures quickly 25 nm longer wavelengths
  16. 16. New Fluorescent Proteins  New approach for monomeric red fluorescent proteins  Replacing N terminus of mRFP1 with GFP  Adding C terminus of GFP to mRFP1
  17. 17. New Fluorescent Proteins  All variants are brighter than mRFP1 (except mHoneydew, mBanana, mTangerine)  mOrange is the brightest but sensitive to pH.  mCherry is the most photostable
  18. 18. New Fluorescent Proteins  Protein lifetimes and turnover rates  First little initial fluorescence with excitation wavelength Then high fluorescence with different wavelength  PA-GFP, Kaede, KFP1
  19. 19. New Fluorescent Proteins  PA-GFP developed from GFP increase in fluorescence when illuminated at 413 nm  Kaede identified from T. geoffroyi converted from a green to a red fluorescent protein by irridation with 350-400 nm  KFP1 from Anemonia sulcata
  20. 20. Summary  We use the fluorescence proteins to see changes in cells  Fluorescence proteins have common properties  Improving fluorescence proteins for better imaging  Examples of new fluorescent proteins
  21. 21. References  Lippincott-Schwartz, J., et al., 2003. Development adn Use of Fluorescent Protein Markers in Living Cells. Science, 300(87), p.87-91  Miyawaki, A., Sawano, A., Kogure, T., 2003. Lightening up cells: labelling proteins with fluorophores. Nature Cell Biology, 5, p.S1-S7  Patterson, G.H., 2004. A new harvest of fluorsecent proteins. Nature Biotechnology, 22(12), p.1524-1525  Rizzo, M.A., Springer, G.H., Granada, B., Piston, D.W., 2004. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnology, 22(4), p.445-449  Sekar, R.B., Periasamy, A., 2003. Fluorescence resonance energy transefer (FRET) microscopy imaging of live cell protein locations. The Journal of Cell Biology, 160(5), p.629-633  Shaner, N.C., Steinbach, P.A., Tsien, R.Y., 2005. A guide to choosing fluorescent proteins. Nature Biotechnology, 2(12), p.905-909  Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N.G., Palmer, A.C., Tsien, R.Y., 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteins. Nature Biotechnology, 22(12), p.1567-1572  Zhang, J., Campbell, R.E., Ting, A.Y., Tsien, R.Y., 2002. Creating New Fluorescent Probes for Cell Biology. Nature, 3, p.906-918
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