Fluorescent Protein Guide: Subcellular Localization

Background

The subcellular localization of a protein is often tied to its function, so it's important to determine where your protein of interest resides. There are a variety of methods for determining subcellular localization. One of the most common is to use fluorescence microscopy to compare the localization of your protein of interest with known markers.

Addgene has assembled a collection of fluorescent fusion proteins that are targeted to various organelles or subcellular structures. You should fuse your protein of interest with a fluorescent protein that has a different emission wavelength. For example, if the marker protein is fused to GFP, you may want to fuse your protein to a red fluorescent protein (see this fusion protein guide to find the appropriate backbone). Then, by examining the overlap between your fusion protein and the marker fusion protein using fluorescence microscopy, you can assess whether your protein is targeted to the same structures as these well-characterized proteins.

General Structures

Browse or search the table below to find markers of general cellular organelles and structures.

Plasmid	Structure	Targeting Gene/Signal	Fluorescent Protein	PI
H2B-mCherry	Chromatin	H2B	mCherry	Benezra
H2B-GFP	Chromatin	H2B	GFP	Wahl
pCAG-mGFP	Membrane	palmitoylation sequence from GAP43	EGFP	Cepko
pPalmitoyl-mTurquoise2	Membrane	palmitoylation sequence from p63	mTurquoise2	Gadella
pmTurquoise2-Mito	Mitochondria	COX8A(1-29)	mTurquoise2	Gadella
pEBFP2-Nuc	Nucleus	NLS	EBFP2	Campbell
pmTurquoise2-H2A	Nucleus	H2A	mTurquoise2	Gadella
CAV1-mCherry	Caveolae	Cav1	mCherry	Helenius
pABCb10-GFP	Mitochondria	ABCb10	GFP	Shirihai
mCherry-Mito-7	Mitochondria	Mitochondrialtargeting sequence(COX8A)	mCherry*	Davidson
mito-BFP	Mitochondria	mitochondrial targeting signal (COX4)	TagBFP	Voeltz
pmTurquoise2-NES	Non-nucleus	Nuclear Export Sequence	mTurquoise2	Gadella
pQC NLS mCherry IX	Nucleus	NLS	mCherry	Cepko
pmTurquoise2-Peroxi	Peroxisomes	Peroxisomal Targeting Sequence	mTurquoise2	Gadella
GFP-Mff	Mitochondria-Outer Membrane	Mff	acGFP1	Voeltz
mCherry-LaminB1-10	Nuclear Envelope	LaminB1	mCherry*	Davidson
mCherry-LaminA-C-18	Nuclear Envelope	LaminA1	mCherry*	Davidson
pEGFP-C1-Fibrillarin	Nucleolus	Fibrillarin	EGFP	Huang
pmKalama1-Nuc	Nucleus	NLS	mKamala1	Campbell
pQC NLS YFP IX	Nucleus	NLS	YFP	Cepko
pcDNA3.1-kappa-myc-dL5-2xG4S-TMst	Cell surface (mammalian)	PDGFR-derived	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez
pH2B-miRFP703	Chromatin	H2B	miRFP703	Verkhusha
pcDNA3.1-KozATG-dL5-2XG4S-mCer3	Cytosol, nucleoplasm	None	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez
cfSGFP2-N	Extracellular milieu	cfSGFP2-N	EGFP	Wada
Lck-GFP	Membrane	palmitoylation sequence from Lck	EGFP	Green
pcDNA3.1-COXIV-COX8-dL5-2XG4S-mCer3	Mitochondria	COX IV-derived (1-22 aa) import sequence and COX VIII signal peptide	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez
pMito-miRFP703	Mitochondria	COX8A	miRFP703	Verkhusha
mCherry-TOMM20-N-10	Mitochondria-Outer Membrane	TOMM20	mCherry*	Davidson
pmScarlet-i_NES_C1	Non-nucleus	Nuclear Export Sequence	mScarlet-I	Gadella
pmScarlet-H_NES_C1	Non-nucleus	Nuclear Export Sequence	mScarlet-H	Gadella
pmScarlet_NES_C1	Non-nucleus	Nuclear Export Sequence	mScarlet	Gadella
pcDNA3.1-NLS-myc-dL5-2xG4S-mCer3	Nucleus	NLS (from Mak16p protein)	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez
pmScarlet-i_H2A_C1	Nucleus	H2A	mScarlet-I	Gadella
pmScarlet-H_H2A_C1	Nucleus	H2A	mScarlet-H	Gadella
pmScarlet_H2A_C1	Nucleus	H2A	mScarlet	Gadella
pTDpelB-C_sfYFPTwinStrep	Periplasmic space	PelB signal sequence	YFP	Dammeyer
pmScarlet-I_peroxisome_C1	Peroxisome	SRL	mScarlet-I	Gadella
pmScarlet-H_peroxisome_C1	Peroxisome	SRL	mScarlet-H	Gadella
pmScarlet_peroxisome_C1	Peroxisome	SRL	mScarlet	Gadella
mCherry-Peroxisomes-2	Peroxisomes	Peroximal Targeting Signal 1 (PTS1)	mCherry*	Davidson
pcDNA3.1-KozATG-myc-dL5-2XG4S-mCer3-SKL	Peroxisomes	SKL tripeptide, peroxisome transport signal	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez

*Fusions to other FPs can be found by searching the complete Michael Davidson Fluorescent Protein Collection.

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Cytoskeletal Elements

Browse or search the table below to find markers of cytoskeletal elements.

Plasmid	Structure	Targeting Gene/Signal	Fluorescent Protein	PI
pCAG-mGFP-Actin	Actin Filaments	beta-actin	EGFP	Yasuda
pLifeAct-mTurquoise2	Actin Filaments	LifeAct	mTurquoise2	Gadella
GFP-UtrCH	Actin Filaments	utrophin (aa# 1-261)	GFP	Bement
pIRESneo-EGFP-alpha Tubulin	Microtubules	alpha-tubulin	EGFP	Wadsworth
pmTurquoise2-Tubulin	Microtubules	alpha-tubulin	mTurquoise2	Gadella
mCh-alpha-tubulin	Microtubules	alpha-tubulin	mCherry	Voeltz
pPAmCherry-b-actin	Actin Filaments	beta-actin	PAmCherry1	Verkhusha
mCherry-Lifeact-7	Actin Filaments	LifeAct	mCherry*	Davidson
pDEST/LIfeAct-mCherry-N1	Actin Filaments	LifeAct	mCherry	Shaw
pEGFP Centrin2	Centrioles (dependent on cell cycle)	Centrin-2	EGFP	Nigg
pPAmCherry-a-tubulin	Microtubules	alpha-tubulin	PAmCherry	Verkhusha
EMTB-3XGFP	Microtubules	ensconsin	GFP	Bement
EMTB-mCherry	Microtubules	ensconsin	mCherry	Bement
pLifeAct_mScarlet_N1	Actin Filaments	LifeAct	mScarlet	Gadella
pEGFP PICH	Centromeres (dependent on cell cycle)	PICH	EGFP	Nigg
pEGFP-mNFM	Intermediate Filaments (neuronal)	Nefm	EGFP	Brown
pLifeAct-miRFP703	Actin Filaments	LifeAct	miRFP703	Verkhusha
pLifeAct_mScarlet-i_N1	Actin Filaments	LifeAct	mScarlet-I	Gadella
pLifeAct_mScarlet-H_N1	Actin Filaments	LifeAct	mScarlet-H	Gadella
pEGFP Cep170 C-term	Centrioles (dependent on cell cycle)	Cep170	EGFP	Nigg
mEmerald-PLK1-N-16	Centrosome	Plk1	mEmerald*	Davidson
pVimentin-miRFP703	Intermediate Filaments (vimentin)	vimentin	miRFP703	Verkhusha
pKeratin-miRFP703	Intermediate Filaments (epithelial)	Keratin	miRFP703	Verkhusha
mCherry-Tubulin-C-18	Microtubules	alpha-tubulin	mCherry*	Davidson
pDEST/N1-hEB1-GFP	Microtubules	EB1	GFP	Shaw
pmiRFP703-Tubulin	Microtubules	alpha-tubulin	miRFP703	Verkhusha
pEB3-miRFP703	Microtubules	EB3	miRFP703	Verkhusha
pmScarlet-i_alphaTubulin_C1	Microtubules	alpha-tubulin	mScarlet-I	Gadella
pmScarlet-H_alphaTubulin_C1	Microtubules	alpha-tubulin	mScarlet-H	Gadella
pmScarlet_alphaTubulin_C1	Microtubules	alpha-tubulin	mScarlet	Gadella
pEGFP-rNFM	Intermediate Filaments (neuronal)	Nefm	EGFP	Brown

*Fusions to other FPs can be found by searching the complete Michael Davidson Fluorescent Protein Collection.

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Cell Junctions

Browse or search the table below to find markers of cellular junctions.

Plasmid	Structure	Targeting Gene/Signal	Fluorescent Protein	PI
pDEST/hCx43-EGFP-N1	Gap Junctions	Connexin 43	EGFP	Shaw
pDEST-mCherry-GJA1-20k-N1	Gap Junctions	Connexin 43 / GJA1-20k	mCherry	Shaw
mCherry-Cx43-7	Gap Junctions	Connexin 43 / GJA1-20k	mCherry*	Davidson
E-cadherin-GFP	Adherens junctions	E-cadherin	GFP	Stow
Paxillin-pEGFP	Focal adhesions	paxillin	GFP	Horwitz
pPaxillin-PSmOrange	Focal adhesions	paxillin	PSmOrange	Verkhusha
mEmerald-Paxillin-22	Focal Adhesions	paxillin	mEmerald*	Davidson
mApple-VE-Cadherin-N-10	Tight Junctions	VE-Cadherin	mApple	Davidson
pZyxin-miRFP703	Focal adhesions	Zyxin	miRFP703	Verkhusha

*Fusions to other FPs can be found by searching the complete Michael Davidson Fluorescent Protein Collection.

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Secretory Pathway

Browse or search the table below to find markers of secretory pathway components.

Plasmid	Structure	Targeting Gene/Signal	Fluorescent Protein	PI
pEF.myc.ER-E2-Crimson	Endoplasmic Reticulum	ER retention signal	E2-Crimson	Glick
pmTurquoise2-ER	Endoplasmic Reticulum	ER retention signal	mTurquoise2	Gadella
mCh-Sec61 beta	Endoplasmic Reticulum	Sec61	mCherry	Voeltz
BFP-Sec61 beta	Endoplasmic Reticulum	Sec61	BFP	Voeltz
BFP-KDEL	Endoplasmic Reticulum	BiP ER signal sequence	BFP	Voeltz
Rtn4a-GFP	Endoplasmic Reticulum	Rtn4a	AcGFP	Voeltz
ERoxBFP	Endoplasmic Reticulum	ER retention signal	oxBFP	Snapp
ERmoxGFP	Endoplasmic Reticulum	ER retention signal	moxGFP	Snapp
GolgiCFP	Golgi	eNOS(1-33)	CFP	Newton
pmTurquoise2-Golgi	Golgi	B4GALT1(1-61)	mTurquoise2	Gadella
GalToxBFP	Golgi	GalT signal anchor transmembrane domain	oxBFP	Snapp
Clathrin-LCa-EYFP	Clathrin-coated vesicles	Clathrin, light polypeptide	EYFP	Zhuang
mRFP-Clc	Clathrin-coated vesicles	Clathrin, light polypeptide	mRFP	Helenius
mEmerald-Clathrin-15	Clathrin-coated vesicles	Clathrin	mEmerald*	Davidson
DsRed-Rab5 WT	Early endosomes	RAB5A	DsRed2	Pagano
mRFP-Rab5	Early endosomes	RAB5A	mRFP	Helenius
mCh-Rab5	Early endosomes	Rab5	mCherry	Voeltz
BFP-Rab5	Early endosomes	Rab5	BFP	Voeltz
GFP-Rab5B	Early endosomes	Rab5B	AcGFP	Voeltz
GFP-Rab4B	Early endosomes	Rab4B	AcGFP	Voeltz
GFP-rab7 WT	Late endosomes	RAB7	GFP	Pagano
DsRed-rab7 WT	Late endosomes	RAB7	DsRed	Pagano
mCh-Rab7A	Late endosomes	Rab7a	mCherry	Voeltz
GFP-Rab7A	Late endosomes	Rab7a	AcGFP	Voeltz
GFP-rab11 WT	Recycling endosomes	RAB11A	GFP	Pagano
DsRed-rab11 WT	Recycling endosomes	RAB11A	DsRed	Pagano
Lamp1-YFP	Lysosomes	Lamp-1	YFP	Mothes
Lamp1-RFP	Lysosomes	Lamp-1	RFP	Mothes
mCherry-Lysosomes-20	Lysosomes	LAMP1	mCherry*	Davidson
pCMB-AUTr	Autophagosome	LC3(ATG8)	mCherry	Harrison
pEGFP-LC3 (human)	Autophagosome	LC3	EGFP	Finkel
pLAMP1-miRFP703	Lysosomes	LAMP1	miRFP703	Verkhusha
pmiRFP703-Clathrin	Clathrin-coated vesicles	Clathrin	miRFP703	Verkhusha
pmScarlet-i_Giantin_C1	Golgi apparatus	Giantin	mScarlet-I	Gadella
pmScarlet-H_Giantin_C1	Golgi apparatus	Giantin	mScarlet-H	Gadella
pmScarlet_Giantin_C1	Golgi apparatus	Giantin	mScarlet	Gadella
pCytERM_mScarlet-i_N1	ER	CytERM	mScarlet-I	Gadella
pCytERM_mScarlet-H_N1	ER	CytERM	mScarlet-H	Gadella
pCytERM_mScarlet_N1	ER	CytERM	mScarlet	Gadella
pTag-RFP-C-h-Rab11a-c-Myc	Recycling endosomes	Rab11a	TagRFP	Johnson
pTag-BFP-C-h-Rab11a-c-Myc	Recycling endosomes	Rab11a	TagBFP	Johnson
pTag-RFP-C-h-Rab7-c-Myc	Late endosomes	Rab7	TagRFP	Johnson
pTag-BFP-C-h-Rab7-c-Myc	Late endosomes	Rab7	TagBFP	Johnson
pTag-RFP-C-h-Rab5a-c-Myc	Early endosomes	Rab5a	TagRFP	Johnson
pTag-BFP-C-h-Rab5a-c-Myc	Early endosomes	Rab5a	TagBFP	Johnson
pTag-RFP-C-h-Rab4a-c-Myc	Recycling endosomes	Rab4a	TagRFP	Johnson
pTag-BFP-C-h-Rab4a-c-Myc	Recycling endosomes	Rab4a	TagBFP	Johnson
pcDNA3.1-kappa-myc-dL5-2XG4S-mCer3-KDEL	Endoplasmic reticulum (ER)	KDEL	dL5 FAP (fluorogen activating protein); mCerulean3 fusion	Bruchez

*Fusions to other FPs can be found by searching the complete Michael Davidson Fluorescent Protein Collection.

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Do you have suggestions for other plasmids that should be added to this list?

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