HaloTag: a novel protein labeling technology for cell imaging and protein analysis.
We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
Los
Georgyi V
GV
Promega Corporation, 2800 Woods Hollow Rd., Madison, Wisconsin 53711, USA. georgyi.los@promega.com
Encell
Lance P
LP
McDougall
Mark G
MG
Hartzell
Danette D
DD
Karassina
Natasha
N
Zimprich
Chad
C
Wood
Monika G
MG
Learish
Randy
R
Ohana
Rachel Friedman
RF
Urh
Marjeta
M
Simpson
Dan
D
Mendez
Jacqui
J
Zimmerman
Kris
K
Otto
Paul
P
Vidugiris
Gediminas
G
Zhu
Ji
J
Darzins
Aldis
A
Klaubert
Dieter H
DH
Bulleit
Robert F
RF
Wood
Keith V
KV
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D002477:Cells / Q000166:cytology* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry*; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D006843:Hydrocarbons, Chlorinated; D008164:Luminescent Proteins; D016328:NF-kappa B; D011506:Proteins; D004247:DNA