Method for determining activity of cell cycle regulatory...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase

Reexamination Certificate

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C435S004000, C435S007100

Reexamination Certificate

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07338774

ABSTRACT:
A method for determining the activity of a cell cycle regulatory factor comprising the steps of:preparing a sample for measuring a cyclin-dependent kinase/cyclin complex from living cells;reacting adenosine 5′-O-(3-thiotriphosphate) (ATP-γS) with a substrate for the cyclin-dependent kinase in presence of the sample in order to introduce a monothiophosphate group into a serine or threonine residue of the substrate;labeling the substrate by coupling a labeling fluorophore or a labeling enzyme with a sulfur atom of the introduced monothiophosphate group;measuring the amount of fluorescence from the labeling fluorophore labeling the substrate, or reacting the labeling enzyme labeling the substrate with a substance which generates an optically detectable product by reaction with the labeling enzyme and optically measuring the amount of the generated product; andcalculating the activity of the cyclin-dependent kinase from the measured amount of fluorescence or the measured amount of the generated product with reference to a pre-produced reference curve.

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