Dina N Kovarik MS PhD Digital World Biology Updated April 24 2015 Fluorescent Proteins are Valuable Tools Locate proteins in the cell Track the migration of cells Reporter of expression Sister centromeres ID: 286515
Download Presentation The PPT/PDF document "Using and Analyzing Fluorescent Proteins" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Using and Analyzing Fluorescent Proteins
Dina N Kovarik, MS, PhD. Digital World Biology. Updated
April 24, 2015Slide2
Fluorescent Proteins are Valuable Tools
Locate proteins in the cell
Track the migration of cellsReporter 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.jpgSlide3
Mitochondria and Neuronal Injury
Source: Simon Frasier University, Biological Sciences. 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. Slide4
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-biologyThe 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.Slide5
Using Fluorescent Proteins
Source: “Fluorescent Proteins.”
Scholarpedia
.
http://www.scholarpedia.org/article/Fluorescent_proteins
. Adapted from
Ai et al. 2008.Slide6
Source
: Applications in Confocal
Microscopy: The Fluorescent Protein Color
Palette. Olympus Confocal Microscopy.
http
://
www.olympusconfocal.com/applications/fpcolorpalette.html Slide7
Multicolor Labeling of Living HeLa Cell
Blue
: Nucleus (histones)
Green
: Cytoskeleton (actin)
Yellow
:
M
itochondriaOrange: Golgi apparatus Red: focal adhesions at the cell membraneSource: 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.Slide8
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.Slide9
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 proteinsDiversity 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… Slide10
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 GFPSlide11
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=174
2Q57
4AR7
2Y0G
1HUY 2H5O2H5Q3M24PDB IDSlide12
Research Questions
Is red fluorescent protein (RFP) related to its famous cousin, GFP?
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).