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AssigneeThe Regents of the University of California
InventorsRoger Y. Tsien, Susan S. Taylor, Stephen R. Adams, Ying Ji
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The invention provides labeled proteins suitable for determining the presence of cAMP, other second messengers and organic molecules. The proteins are separately labeled with fluorochromes which, when in close spatial proximity, preferably, less than about 6 nm, interact through the transfer of energy from one fluorochrome to the other. A composition of matter, (S.sub.1.A).sub.n1 (S.sub.2.D).sub.n2 is provided wherein S.sub.1 and S.sub.2 are two proteins which are associated in one state and substantially disassociated in another, the equilibrium between which is controlled by the free concentration of an analyte, and A and D are fluorochromes, the emission wavelength of fluorochrome D overlapping the excitation wavelength of fluorochrome A and the distance between A and D being in sufficiently close proximity to allow the radiationless transfer of energy between the fluorochromes. A and D can be selected from a variety of acceptor, donor pairs such as fluorescein and tetramethylrhodamine and derivatives thereof. The concentration of analytes such as cAMP in a sample can be determined by contacting the sample with (S.sub.1.A).sub.n1 (S.sub.2.D).sub.n2 providing energy near the excitation wavelength of D and measuring the fluorescence of A or D, the concentration of cAMP and other said analytes being determined by the ratio of emission of D to the emission of A, previously calibrated with reference solutions of known analyte concentration.
A composition of matter, comprising (S.sub.1.A).sub.n1 and (S.sub.2.D).sub.n2, wherein S.sub.1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S.sub.2 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said composition.
A composition of matter, comprising (S.sub.1.A).sub.n1 and (S.sub.2.D).sub.n2, wherein S.sub.1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and S.sub.2 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, and wherein n1 and n2 represent the stoichiometry of said subunits in said composition.
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising:
A method of determining the concentration of adenosine 3', 5'-cyclic monophosphate in a sample comprising:
A method of preparing a labeled complex comprising (S.sub.1.A) and (S.sub.2.D), wherein S.sub.1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S.sub.2 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, said method comprising:
A method of preparing a labeled complex comprising (S.sub.1.A) and (S.sub.2.D), wherein S.sub.1 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S.sub.2 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, wherein a first complex comprising S.sub.1 and S.sub.2 is attached to fluorochrome A, and a second complex comprising S.sub.1 and S.sub.2 is attached to fluorochrome D;
A method of preparing a labeled complex comprising (S.sub.1.A) and (S.sub.2.D), wherein S.sub.1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S.sub.2 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlapping the excitation wavelength of A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, said method comprising:
A method of preparing a labeled complex comprising (S.sub.1 .A) and (S.sub.2.D), wherein S.sub.1 is the catalytic subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase and S.sub.2 is the regulatory subunit of adenosine 3', 5'-cyclic monophosphate dependent protein kinase, and wherein D and A are different fluorochromes the emission wavelength of D overlaps the excitation wavelength of the other A, and D and A in said complex are in sufficiently close proximity to allow radiationless transfer of energy between the fluorochromes, wherein a first complex comprising S.sub.1 and S.sub.2 is attached to fluorochrome A, and a second complex comprising S.sub.1 and S.sub.2 is attached to fluorochrome D;
Dependencies are inferred from the claim text.