Fluorescent Proteins: 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.

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
	pLenti-EB1-EGFP	Microtubules	EB1	EGFP	Takemaru
	pEGFP-rNFM	Intermediate Filaments (neuronal)	Nefm	EGFP	Brown
	GFP-hChibby1	Mother Centrioles / Ciliary Base	Chibby1	GFP	Takemaru
	pLenti-EGFP-hChibby1	Mother Centrioles / Ciliary Base	Chibby1	EGFP	Takemaru
	GFP-hCCDC11	Centriolar satellites	CCDC11	EGFP	Takemaru

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

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.

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.