Fluorescent polarization assays involving multivalent metal ions and systems
First Claim
1. A method of determining whether a compound is phosphorylated, the compound comprising a fluorescent label, the method comprising:
- providing the compound in a mixture with a binding component, wherein the binding component comprises multivalent metal ions and is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the compound, when the binding component binds the compound; and
detecting whether the binding component binds to the compound by monitoring the amount of polarized fluorescence emitted from the mixture, binding of the binding component to the compound being indicative that the compound is phosphorylated.
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Abstract
Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.
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Citations
19 Claims
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1. A method of determining whether a compound is phosphorylated, the compound comprising a fluorescent label, the method comprising:
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providing the compound in a mixture with a binding component, wherein the binding component comprises multivalent metal ions and is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the compound, when the binding component binds the compound; and
detecting whether the binding component binds to the compound by monitoring the amount of polarized fluorescence emitted from the mixture, binding of the binding component to the compound being indicative that the compound is phosphorylated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. An assay system, comprising:
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a first reaction vessel having disposed therein a first reaction mixture comprising a phosphorylated first fluorescent compound, and a binding component, wherein the binding component comprises multivalent metal ions and is sufficiently large to induce a shift in an amount of polarized fluorescence emitted by the compound when bound by the binding component; and
a fluorescence polarization detector positioned in sensory communication with the first mixture in the reaction vessel. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19)
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Specification