Screening using polarization anisotropy in FRET emissions
First Claim
1. A method for detecting specific binding between a first chemical entity and a second chemical entity, the method comprising:
- immobilizing the first chemical entity in association with a first fluorophore;
allowing the second chemical entity to bind with the immobilized first chemical entity, wherein the second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore;
exposing the bound chemical entities to radiation at an excitation frequency for either the first or the second fluorophore; and
measuring polarization anisotropy of a FRET fluorescent signal from the bound chemical entities to detect specific binding between the first and second chemical entities,wherein the polarization anisotropy is measured at a first wavelength and a second wavelength.
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Abstract
Methods and apparatus are described for detecting specific binding between first and second chemical entities. The first chemical entity in association with a first fluorophore is immobilized. The second chemical entity is allowed to bind with the immobilized first chemical entity. The second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore. The bound chemical entities are exposed to radiation at an excitation frequency for either the first or the second fluorophore, and polarization anisotropy of a FRET fluorescent signal from the bound chemical entities is measured to detect specific binding between the first and second chemical entities. Techniques are also disclosed for detecting whether a FRET interaction is occurring between a first chemical entity including a donor fluorophore and a second chemical entity including an acceptor fluorophore, using simultaneous anisotropy measurements at the wavelengths of the donor and acceptor fluorophores.
12 Citations
36 Claims
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1. A method for detecting specific binding between a first chemical entity and a second chemical entity, the method comprising:
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immobilizing the first chemical entity in association with a first fluorophore; allowing the second chemical entity to bind with the immobilized first chemical entity, wherein the second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore; exposing the bound chemical entities to radiation at an excitation frequency for either the first or the second fluorophore; and measuring polarization anisotropy of a FRET fluorescent signal from the bound chemical entities to detect specific binding between the first and second chemical entities, wherein the polarization anisotropy is measured at a first wavelength and a second wavelength. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method for detecting whether a FRET interaction is occurring between a first chemical entity in association with a donor fluorophore and a second chemical entity in association with an acceptor fluorophore, the method comprising:
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exposing at least one of the fluorophores to radiation at an excitation wavelength for the donor fluorophore; measuring a polarization anisotropy of a fluorescent signal at an emission wavelength for the donor fluorophore and at an emission wavelength for the acceptor fluorophore; and comparing the measured anisotropies to determine whether a FRET interaction is occurring between the first and second chemical entities. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A method for detecting whether a FRET interaction is occurring between a first chemical entity in association with a donor fluorophore and a second chemical entity in association with an acceptor fluorophore, the method comprising:
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exposing a region containing the fluorophores to radiation at an excitation wavelength for the donor fluorophore; measuring a polarization anisotropy of a fluorescent signal at an emission wavelength for the acceptor fluorophore; measuring a polarization anisotropy at the wavelength of a reference signal in the regions containing the fluorophores; and comparing the measured anisotropies to determine whether a FRET interaction is occurring between the first and second chemical entities. - View Dependent Claims (36)
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Specification