Using and Analyzing Fluorescent Proteins Dina N Kovarik, MS, PhD. Digital World Biology. Updated...
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![Page 1: Using and Analyzing Fluorescent Proteins Dina N Kovarik, MS, PhD. Digital World Biology. Updated September 11, 2014.](https://reader036.fdocuments.in/reader036/viewer/2022062515/56649f3f5503460f94c60253/html5/thumbnails/1.jpg)
Using and Analyzing Fluorescent Proteins
Dina N Kovarik, MS, PhD. Digital World Biology. Updated September 11, 2014
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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: http://mct.aacrjournals.org/content/2/5.cover-expansion
Mice expressing GFP under UV light (left & right), compared to normal mouse (center). Source: Wikipedia.
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Mitochondria and Neuronal Injury
http://www.sfu.ca/rintoul-lab/research.html
Fluorescence micrographs (low and high magnification) of a neuron co-transfected with mitochondrially-targeted yellow fluorescent protein and cytoplasmic cyan fluorescent protein.
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In planta Cytometry
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.
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Using Fluorescent Proteins
http://www.scholarpedia.org/article/Fluorescent_proteins
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http://www.olympusconfocal.com/applications/fpcolorpalette.html
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Multicolor Labeling of Living HeLa Cell
Blue: histone (nucleus). Green: actin (cytoskeleton). Yellow: mitochondria. Orange: golgi. Red: focal adhesions (cell membrane, signal transduction).
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Bioluminescence of the crystal jellyfish, Aequorea victoria
Source: http://www.conncoll.edu/ccacad/zimmer/GFP-ww/shimomura.html
238 amino acid proteins. GFP ribbon diagram. From PDB 1EMA.
Source: Wikipedia
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Rainbow of Fluorescent Proteins
Source: Wikipedia. http://en.wikipedia.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…
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Spectral Diversity of Monomeric FPs
Chudakov et al, 2010. http://physrev.physiology.org/content/90/3/1103
Emission Wavelength (nm)
Brig
htne
ss R
elati
ve to
GFP
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http://www.rcsb.org/pdb/101/motm.do?momID=174
2Q57
4AR7
2Y0G
1HUY
2H5O
2H5Q3M24
PDB ID
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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).