Fluorescent Protein Guide: FRET

Background

Förster resonance energy transfer, or FRET, is a process by which energy is non-radiatively transferred from an excited donor fluorophore to an acceptor. Since the transfer of energy does not occur by emission of a photon, the acceptor molecule is not required to be fluorescent. A variety of organic dyes and fluorescent proteins have been developed for use as donor and acceptor pairs in FRET experiments. The efficiency of the energy transfer is measured using fluorescence microscopy by exciting the donor and measuring the emission of the acceptor.

For a given donor-acceptor pair, the FRET efficiency strongly depends on the distance between the donor and acceptor molecules and can therefore be used to calculate the distances between the donor and acceptor. FRET is often used to study (1) protein-protein interactions where each protein is separately fused to a donor or acceptor molecule (also referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the donor and acceptor are both fused to the same protein (also referred to as intramolecular or unimolecular FRET). Fluorescent proteins can be used as donor and/or acceptor molecules in either of these types of studies

Addgene has a collection of plasmids suitable for creating individual fluorescently tagged proteins to study protein-protein interactions and a series of FRET standards.

Pre-constructed fluorescent biosensors targeting small molecules or specific genes are also available. Custom biosensors can also be constructed using the cpFRET kit from the Pertz laboratory.

Empty Vectors Encoding Fluorescent Proteins for FRET

The following plasmids can be used to create a fluorescent fusion protein with your gene of interest and the listed fluorescent protein.

Plasmid	Color	Expression	Description
pPROEX Aqua	Cyan	Bacterial	Expresses Aquamarine with N-terminal His tag
pAquaN1	Cyan	Mammalian	Expresses mammalian optimized Aquamarine
mCerulean N1	Cyan	Mammalian	Express a gene of interest fused to the N-terminus of monomeric Cerulean
mCerulean C1	Cyan	Mammalian	Express a gene of interest fused to the C-terminus of monomeric Cerulean
mTurquoise2	Cyan	Mammalian	Constructs to target mTurquoise2 to various subcellular compartments
pCEP4CyPet-MAMM	Cyan	Mammalian	Expresses mammalian optimized CyPet
pCyPet-His	Cyan	Bacterial	Expresses CyPet with C-terminal His tag
SCFP3A	Cyan	Mammalian	Express a gene of interest fused to the C-terminus of SCFP3A
Amber N1	Yellow	Mammalian	Express a gene of interest fused to the N-terminus of Amber
Amber C1	Yellow	Mammalian	Express a gene of interest fused to the C-terminus of Amber
mVenus N1	Yellow	Mammalian	Express a gene of interest fused to the N-terminus of monomeric Venus
mVenus C1	Yellow	Mammalian	Express a gene of interest fused to the C-terminus of monomeric Venus
pCEP4YPet-MAMM	Yellow	Mammalian	Expresses mammalian optimized YPet
pYPet-His	Yellow	Bacterial	Expresses YPet with C-terminal His tag
SYFP2	Yellow	Mammalian	Express a gene of interest fused to the C-terminus of SYFP2
Clover	Green	Mammalian	Expresses Clover (a GFP variant) commonly used with mRuby2
pLSSmOrange-N1	Orange	Mammalian	Express a gene of interest fused to the N-terminus of LSSmOrange
pLSSmOrange-C1	Orange	Mammalian	Express a gene of interest fused to the C-terminus of LSSmOrange
mRuby2	Red	Mammalian	Expresses mRuby2 (a RFP variant) commonly used with Clover
pGWF1	Cyan & Yellow	Bacterial	Gateway-compatible vector to express a gene of interest fused between ECFP and Venus

FRET Reference Standards

The following plasmids have well-characterized FRET efficiency values and were developed to be used as FRET standards to calibrate the numerous and disparate methods used to measure FRET.

Plasmid	FRET Pair
C5V	Cerulean attached to Venus via a 5 amino acid linker
C17V	Cerulean attached to Venus via a 17 amino acid linker
C32V	Cerulean attached to Venus via a 32 amino acid linker
C5A	Cerulean attached to Amber via a 5 amino acid linker
C17A	Cerulean attached to Amber via a 17 amino acid linker
C32A	Cerulean attached to Amber via a 32 amino acid linker
mGFP-10-sREACh-N3	Monomeric EGFP attached to super-REACh via a 10 amino acid linker
ACA	Heterotrimeric construct consisting of Amber-5aa linker-Cerulean-6aa linker-Amber
ACV	Heterotrimeric construct consisting of Amber-5aa linker-Cerulean-6aa linker-Venus
VCA	Heterotrimeric construct consisting of Venus-5aa linker-Cerulean-6aa linker-Amber
VCV	Heterotrimeric construct consisting of Venus-5aa linker-Cerulean-6aa linker-Venus
ACAV	Heterotetrameric construct consisting of Amber-5aa linker-Cerulean-5aa linker-Amber-6aa linker-Venus
ACVA	Heterotetrameric construct consisting of Amber-5aa linker-Cerulean-5aa linker-Venus-6aa linker-Amber
VCAA	Heterotetrameric construct consisting of Venus-5aa linker-Cerulean-5aa linker-Amber-6aa linker-Amber
VCVV	Heterotetrameric construct consisting of Venus-5aa linker-Cerulean-5aa linker-Venus-6aa linker-Venus
V5V	Anisotropy/brightness standard consisting of two Venus fluorescent proteins connected via a 5 amino acid linker
V17V	Anisotropy/brightness standard consisting of two Venus fluorescent proteins connected via a 17 amino acid linker
V32V	Anisotropy/brightness standard consisting of two Venus fluorescent proteins connected via a 32 amino acid linker
VVV	Anisotropy/brightness standard consisting of three Venus fluorescent proteins connected via a 5 and 6 amino acid linker, respectively
VVVV	Anisotropy/brightness standard consisting of four Venus fluorescent proteins connected via a 5, 5 and 6 amino acid linker, respectively
VVVVV	Anisotropy/brightness standard consisting of five Venus fluorescent proteins connected via 5 amino acid linkers
VVVVVV	Anisotropy/brightness standard consisting of six Venus fluorescent proteins connected via 5 amino acid linkers
pET28CLY1	Peptide linker standard consisting of ECFP and EYFP connected by 1 flexible glycine- and serine-containing peptide linker (GGSGGS) repeat
pET28CLY2	Peptide linker standard consisting of ECFP and EYFP connected by 2 GGSGGS repeats
pET28CLY3	Peptide linker standard consisting of ECFP and EYFP connected by 3 GGSGGS repeats
pET28CLY4	Peptide linker standard consisting of ECFP and EYFP connected by 4 GGSGGS repeats
pET28CLY5	Peptide linker standard consisting of ECFP and EYFP connected by 5 GGSGGS repeats
pET28CLY6	Peptide linker standard consisting of ECFP and EYFP connected by 6 GGSGGS repeats
pET28CLY7	Peptide linker standard consisting of ECFP and EYFP connected by 7 GGSGGS repeats
pET28CLY8	Peptide linker standard consisting of ECFP and EYFP connected by 8 GGSGGS repeats
pET28CLY9	Peptide linker standard consisting of ECFP and EYFP connected by 9 GGSGGS repeats
pmVenus(L68V)-mTurquoise2	Brightness standard used as positive control to characterize mTurquoise2
pmTurquoise2-T2A-Venus(L68V)	Brightness standard used a negative control (no FRET) with pmVenus(L68V)-mTurquoise2