Versatile Strategy for
Biochemical, Electrochemical
and Immunoarray Detection of Protein Phosphorylations
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Abstract
Protein kinases catalyze the phosphorylation of cellular
proteins
involved in the regulation of many cellular processes and have emerged
as promising targets for the treatment of several diseases. Conventional
assays to monitor protein kinase activity are limited because they
typically rely on transfer of radioactive phosphate or phospho-specific
antibodies that recognize specific substrates or sequence motifs.
To overcome the limitations of conventional assays, we have developed
a versatile approach based on transfer of ferrocene-phosphate that
can be readily monitored using electrochemical detection or detection
with antiferrocene antibodies in an immunoarray format. This assay
is readily adapted to multiplex arrays and can be employed for monitoring
kinase activity in complex mixtures and for kinase inhibitor profiling