What is fluorescence Use of fluorophores Protein synthesis Protein folding
Stokes Shift - is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
Tracking - Qualitative
- Quantitative
- DNA, protein, lipids, ions, signaling molecules
- Nearly as sensitive as radioactivity, and a lot safer
FITC 488 525 PE 488 575 APC 630 650 PerCP™ 488 680 Cascade Blue 360 450 Coumerin-phalloidin 350 450 Texas Red™ 610 630 Tetramethylrhodamine-amines 550 575 CY3 (indotrimethinecyanines) 540 575 CY5 (indopentamethinecyanines) 640 670
Resonance Energy Transfer
Chemically add Chemically add - Limited to small molecules
Biosynthetically incorporate - Genetically engineer
- GFP and derivatives large (>20kD)
Stages Components How can the system be altered to incorporate unnatural amino acids
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