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.
1 Assignment
0 Petitions
Accused Products
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.
-
Citations
37 Claims
-
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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
-
-
24. 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:
-
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 (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
-
-
36. 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:
-
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 (37)
-
Specification