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).