Fluorescence Lifetime and Fluorescence Assays
OwnerAlexander Gray, Michael John Paterson +1inventors
InventorsAlexander Gray, Michael John Paterson, Dmitry Gakamsky
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Abstract
The invention provides a method for determining a degree of phosphorylation of a substrate, for example a peptide substrate, using a fluorescence probe that acts alone or with another material and has a lifetime that varies when in proximity to a phosphate, the method comprising: causing the fluorescence probe to fluoresce; measuring a time response of the fluorescence, and analysing the fluorescence time response to identify a fluorescence component having a lifetime associated with phosphorylated substrate and a fluorescence component having a lifetime associated with un-phosphorylated substrate.
Claims48
A method for determining a degree of phosphorylation of a substrate, using a fluorescence probe that acts alone or with another material and has a lifetime that varies when in proximity to a phosphate, the method comprising:
- causing the fluorescence probe to fluoresce;
- measuring a time response of the fluorescence, and
- analyzing the fluorescence time response to identify a fluorescence component having a lifetime associated with phosphorylated substrate and a fluorescence component having a lifetime associated with un-phosphorylated substrate.
A method for investigating the phosphorylation of a fluorescently labeled test compound by a kinase, said method comprising contacting said test compound with said kinase and a quenching moiety and measuring the resultant fluorescence, and said quenching moiety quenching fluorescence from the test compound upon phosphorylation of the test compound.
A method for investigating the dephosphorylation of a fluorescently labeled test compound by a phosphatase, wherein said test compound comprises a phosphate moiety, said method comprising contacting said test compound with said phosphatase and a quenching moiety and measuring the resultant fluorescence, and said quenching moiety quenching fluorescence from the test compound.
A method for investigating the activity of a kinase, said method comprising
- (i) exposing the kinase to a test compound in the presence of a complex comprising a fluorescently labeled phosphorylated compound and quenching moiety, wherein said quenching moiety quenches fluorescence of the fluorescent label in said complex;
- and
- (ii) measuring the resultant fluorescence.
A method for investigating the activity of phosphatase, said method comprising
- (i) exposing the phosphatase to a test compound wherein some of said test compound is present in a complex comprising said test compound and quenching moiety, in the presence of a fluorescently labeled phosphorylated compound wherein said quenching moiety quenches fluorescence of the fluorescent label upon complexation of the protein to the fluorescently labeled phosphorylated compound;
- and
- (ii) measuring the resultant fluorescence.
(canceled)
Dependencies are inferred from the claim text.