KINASE ACTIVITY DETECTION METHODS

摘要

A strategy to take advantage of time-resolved luminescence of Ln3+-chelated phosphotyrosine-containing peptides, which facilitate efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally-colored biosensors for two different kinases is provided. The method enables multiplexed detection with high signal to noise in a high-throughput-compatible format and a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies.

主权项

1. A method for detecting the activities of two or more kinases comprising:
a) contacting a first kinase and a second kinase with a first peptide and a second peptide, wherein:
i) the first peptide is a substrate for the first kinase;ii) the second peptide is a substrate for the second kinase;iii) each peptide is associated with a lanthanide;iv) each peptide comprises a group capable of sensitizing the lanthanide that is associated with that peptide; andv) each peptide is linked to a fluorphoreunder conditions such that a first signal associated with the activity of the first kinase and a second signal that is associated with the activity of the second kinase are generated; and
b) detecting the first signal and the second signal.