Using and Analyzing Fluorescent Proteins Dina N Kovarik, MS, PhD. Digital World Biology. Updated...
-
Upload
chrystal-russell -
Category
Documents
-
view
218 -
download
0
Transcript of Using and Analyzing Fluorescent Proteins Dina N Kovarik, MS, PhD. Digital World Biology. Updated...
Using and Analyzing Fluorescent Proteins
Dina N Kovarik, MS, PhD. Digital World Biology. Updated September 11, 2014
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.
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.
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.
Using Fluorescent Proteins
http://www.scholarpedia.org/article/Fluorescent_proteins
http://www.olympusconfocal.com/applications/fpcolorpalette.html
Multicolor Labeling of Living HeLa Cell
Blue: histone (nucleus). Green: actin (cytoskeleton). Yellow: mitochondria. Orange: golgi. Red: focal adhesions (cell membrane, signal transduction).
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
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…
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
http://www.rcsb.org/pdb/101/motm.do?momID=174
2Q57
4AR7
2Y0G
1HUY
2H5O
2H5Q3M24
PDB ID
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).