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Repetitive reversible labeling of proteins at polyhistidine sequences for single-molecule imaging in live cells.

Chemphyschem : a European journal of chemical physics and physical chemistry (2007-04-17)
Emmanuel G Guignet, Jean-Manuel Segura, Ruud Hovius, Horst Vogel
ABSTRACT

Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 647, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 647 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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