PMID- 16018556
OWN - NLM
STAT- MEDLINE
DCOM- 20051107
LR  - 20180607
IS  - 0736-6205 (Print)
IS  - 0736-6205 (Linking)
VI  - 38
IP  - 6
DP  - 2005 Jun
TI  - Peptide tags for labeling membrane proteins in live cells with multiple 
      fluorophores.
PG  - 945-52
AB  - We describe a method to label specific membrane proteins with fluorophores for 
      live imaging. Fusion proteins are generated that incorporate into their 
      extracellular domains short peptide sequences (13-38 amino acids) recognized with 
      high affinity and specificity by protein ligands, alpha-bungarotoxin (BTX), or 
      streptavidin (SA). Many fluorophore- and enzyme-conjugated derivatives of both 
      ligands are commercially available. To demonstrate the general utility of the 
      methods, we tagged a vesicle-associated protein (VAMP2), a receptor tyrosine 
      kinase [muscle-specific kinase (MuSK)], and receptors for three 
      neurotransmitters: acetylcholine (nAChR alpha3), glutamate (mGluR2), and 
      gamma-aminobutyric acid (GABA(A) alpha3). In all cases, we could selectively 
      label surface-exposed proteins without interference from intracellular pools. By 
      successive pulse-labeling with different fluorophore conjugates of a single 
      ligand, we were able to monitor endocytosis of tagged molecules. By combining the 
      two ligands, we could assess co-localization of synaptic components in cells. 
      This strategy for epitope tagging provides a useful adjunct to green fluorescent 
      protein (GFP)-tagging, which fails to distinguish intracellular from 
      extracellular pools, sometimes interferes with protein localization or function, 
      and requires a separate construct for each color.
FAU - McCann, Corey M
AU  - McCann CM
AD  - Harvard University, Cambridge, MA, USA.
FAU - Bareyre, Florence M
AU  - Bareyre FM
FAU - Lichtman, Jeff W
AU  - Lichtman JW
FAU - Sanes, Joshua R
AU  - Sanes JR
LA  - eng
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Biotechniques
JT  - BioTechniques
JID - 8306785
RN  - 0 (DNA Primers)
RN  - 0 (Epitopes)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Membrane Proteins)
RN  - 0 (Peptides)
SB  - IM
MH  - Base Sequence
MH  - Cell Line
MH  - DNA Primers
MH  - Epitopes/chemistry
MH  - Fluorescent Dyes/*chemistry
MH  - Humans
MH  - Membrane Proteins/*chemistry/metabolism
MH  - Peptides/*chemistry
MH  - Transfection
EDAT- 2005/07/16 09:00
MHDA- 2005/11/08 09:00
CRDT- 2005/07/16 09:00
PHST- 2005/07/16 09:00 [pubmed]
PHST- 2005/11/08 09:00 [medline]
PHST- 2005/07/16 09:00 [entrez]
AID - 05386IT02 [pii]
AID - 10.2144/05386IT02 [doi]
PST - ppublish
SO  - Biotechniques. 2005 Jun;38(6):945-52. doi: 10.2144/05386IT02.