1. Using and Analyzing Fluorescent Proteins Dina N Kovarik, MS, PhD. Digital World Biology. Updated April 24, 2015

2. Fluorescent Proteins are Valuable Tools Locate proteins in the cell Track the migration of cells Reporter of expression Sister centromeres (green) mark the attachment of microtubules (red) to sister chromatids (blue). Left: Normal. Right: Drug-treated. Source: Molecular Cancer Therapeutics. May 2003 Cover Image. http://mct.aacrjournals.org/content/2/5.cover-expansion Mice expressing GFP under UV light (left & right), compared to normal mouse (center). Source: "GFP Mice 01" Moen et al. 2012. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation. BMC Cancer. 12:21. doi:10.1186/1471-2407-12-21 PDF. Licensed under CC BY 2.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:GFP_Mice_01.jpg

3. Mitochondria and Neuronal Injury Source: Simon Frasier University, Biological Sciences. http://www.sfu.ca/rintoul-lab/research.html yellow fluorescent protein Fluorescence micrographs (low and high magnification) of a neuron co-transfected with mitochondrially-targeted yellow fluorescent protein and cytoplasmic cyan fluorescent protein.

4. In planta Cytometry Source: Fernan Federici from the Haseloff lab. In “Lighting up plant cells to engineer biology.” April 5, 2012. http://www.cam.ac.uk/research/news/lighting-up-plant-cells-to-engineer-biology The proteins are used to mark and consequently identify specific parts of cells - the nuclei and membrane - mapping the development, position and geometry of the cellular make- up in the living plant tissue.

5. Using Fluorescent Proteins Source: “Fluorescent Proteins.” Scholarpedia. http://www.scholarpedia.org/article/Fluorescent_proteins. Adapted from Ai et al. 2008.http://www.scholarpedia.org/article/Fluorescent_proteins

6. Source: Applications in Confocal Microscopy: The Fluorescent Protein Color Palette. Olympus Confocal Microscopy. http://www.olympusconfocal.com/applications/fpcolorpalette.html http://www.olympusconfocal.com/applications/fpcolorpalette.html

7. Multicolor Labeling of Living HeLa Cell Blue: Nucleus (histones) Green: Cytoskeleton (actin) Yellow Yellow: Mitochondria Orange: Golgi apparatus Red: focal adhesions at the cell membrane Source: Dmitriy M. Chudakov, Mikhail V. Matz, Sergey Lukyanov, Konstantin A. Lukyanov. 2010. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiological Reviews. 95:2. DOI: 10.1152/physrev.00038.2009.

8. Bioluminescence of the Jellyfish Aequorea victoria Source: Marc Zimmer. GFP - Green Fluorescent Protein: History. http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html 238 amino acid proteins. GFP ribbon diagram. From PDB 1EMA. Source: "GFP structure" by Zephyris at the English language Wikipedia. Licensed under CC BY-SA 3.0 via Wikimedia Commons.

9. Rainbow of Fluorescent Proteins Source: "FPbeachTsien" by Nathan Shaner - transferred from en:Image:FPbeachTsien.jpg. Licensed under CC BY-SA 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:FPbeachTsien.jpg “Drawn” with bacteria expressing 8 different fluorescent proteins Diversity of Mutations  Diversity of Colors “mFruits” mBlueberry (Blue Fluorescent Protein, or BFP) mLemon (Yellow Fluorescent Protein, or YFP) mGrape1 (Cyan Fluorescent Protein, or CFP) Many others, all with similarly ‘fruity’ names…

10. Spectral Diversity of Fluorescent Proteins Source: Chudakov et al. (2010). Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiological Reviews. 90:3: 1103-1163. http://physrev.physiology.org/content/90/3/1103 Emission Wavelength (nm) Brightness Relative to GFP

11. Source: Image by David S. Goodsell, RCSB Protein Data Bank (www.rcsb.org) of PDB IDs 3m24, 2q57, 4ar7, 2y0g, 1huy, 2h5o, 2h5q. http://www.rcsb.org/pdb/101/motm.do?momID=174www.rcsb.org 2Q57 4AR7 2Y0G 1HUY 2H5O 2H5Q 3M24 PDB ID

12. Research Questions (1)Is red fluorescent protein (RFP) related to its famous cousin, GFP? (2)What other fluorescent proteins, if any, are closely related to GFP and/or RFP? The cloning and protein purification experiments you have been conducting in the laboratory involve mTomato, also called red fluorescent protein (RFP).