Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells.
A specific protein fluorescent labeling method has been used as a tool for bio-imaging in living cells. We developed a novel system of switching "fluorescent turn on" by the recognition of a fluorescent probe to a hexahistidine-tagged (His-tag) protein. The tetramethyl rhodamine bearing three nitrilotriacetic acids, which was used as a fluorescent probe to target a His-tagged protein, formed a reversible complex with the quencher, (Dabcyl)-conjugated oligohistidines, in the homogeneous solution, causing fluorescence of the fluorophore to be quenched. The complex when applied to living cells (COS-7) expressing His-tagged proteins on the cell surface caused the quencher-conjugated oligohistidines to be dissociated from the complex by specific binding of the fluorescent probe to the tagged protein, resulting in the fluorescent emission. The complex that did not participate in the binding event remained in the quenched state to maintain a low level of background fluorescence.
Comprehensive Research Organization, Waseda University, Tokyo, Japan.
Satoshi
S
Arai
Su-In
SI
Yoon
Atsushi
A
Murata
Masao
M
Takabayashi
Xiaoyu
X
Wu
Yixin
Y
Lu
Shinji
S
Takeoka
Miwako
M
Ozaki
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry*; D002522:Chlorocebus aethiops; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006639:Histidine / Q000032:analysis / Q000737:chemistry; D009942:Organometallic Compounds / Q000032:analysis / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D010455:Peptides; D011506:Proteins; C110932:tetramethyl rhodamine ethyl ester; C033223:polyhistidine; D006639:Histidine; D016650:Fluorescein-5-isothiocyanate