Method for detecting membrane protein internalization

摘要

The instant invention provides for methods for detecting the internalization of a transmembrane protein of interest expressed at the surface of a cell. More specifically, the methods involve (a) labelling the protein of interest with a fluorescent metal complex, the lifetime of which is greater than 0.1 ms, (b) adding to the reaction medium a composition capable of causing the internalization of the protein of interest, (c) adding to the reaction medium (1) a modulating agent which is a fluorescent FRET acceptor compound compatible with the fluorescent metal complex, the final concentration of which in the reaction medium is greater than 10−7M; or (2) a reducing agent, the redox potential of which is less than +0.1 V and preferably between 0.25 and 0.75 V; or (3) an agent which binds specifically, by non-covalent bonding, with the fluorescent metal complex; (d) adding a metal ion which competes with the rare earth so as to form a non-fluorescent metal complex; (d) measuring the luminescence emitted by the reaction medium at the emission wavelength of the fluorescent metal complex and/or at the emission wavelength of the modulating compound when the compound is a fluorescent acceptor compound; and (e) comparing the signal measured in step d) with a reference signal measured on cells having been subjected only to steps a) and c).

主权项

1. A method for detecting the internalization of a transmembrane protein of interest expressed at the surface of a cell which is contained in a reaction medium, comprising:
a) labeling, in said reaction medium, the protein of interest with a fluorescent metal complex comprising a lanthanide or ruthenium, the lifetime of which is greater than 0.1 ms; b) adding to said reaction medium a compound capable of causing the internalization of the protein of interest; c) adding to said reaction medium a modulating agent which is a fluorescent FRET acceptor compound compatible with said fluorescent metal complex, wherein said the acceptor is not conjugated to a compound which can bring said acceptor into proximity with said fluorescent metal complex, and wherein the acceptor freely diffuses in the reaction medium, the final concentration of which in the reaction medium is greater than 10−7 M, and the molecular weight of which is less than 50 kD; d) measuring the signal emitted by said reaction medium at the emission wavelength of the fluorescent metal complex and/or at the emission wavelength of the modulating agent when said modulating agent is a fluorescent acceptor compound; e) comparing the signal measured in step d) with a reference signal measured on cells having been subjected only to steps a), c) and d), a difference in the signal measured in step d) compared with the reference signal being representative of the internalization of the protein of interest.