Visit our Expo - Redox and Inflammation signaling 2012

A Novel Fluorescence-based Kinetic Kinase Assay for Use with Recombinant Protein
and Cell Lysates
M. Li, J.A. Vrana, M. Surby, X-D Qian, C.J. Clark, M. Schults, B. Imperiali, E.M.
Schaefer
Signal Transduction Division, Invitrogen Corporation, Hopkinton, Massachusetts and
Department of Chemistry, Massachusetts Institute of Technology
Protein phosphorylation plays a critical role in signal transduction in normal and disease
states. Recent advances in the development of specific protein kinase inhibitors illustrates the
potential for a new generation of drugs to treat human cancers. We have adapted a novel
fluorescent peptide substrate-based assay for the rapid, homogeneous and sensitive detection
of the activity of a broad range of protein kinases. This assay platform uses the chelationenhanced
fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred
to as Sox), which is incorporated into the substrate peptide. A series of specific Ser/Thr and
Tyr substrate peptides are utilized to measure a range of kinases covering all families of the
kinome. Several key features of this assay include measurement of kinase activity under
optimal kinetics, physiological (mM) ATP, and in real time without the use of radioactive
tracer, beads, antibodies or specialized equipment. Importantly, activity increases result in a
corresponding increase in fluorescence. Because the assay can be performed with a wide
range of ATP concentrations it can be used to select for both ATP-competitive and ATP-noncompetitive
kinase inhibitors. We have applied this system for the analysis of multiple sample
types including the determination of kinase activity of recombinant proteins and using crude
cell lysates in a 96 or 384 well format. This technology, used in conjunction with phosphospecific
antibody based profiling, provides a valuable set of tools for rapidly increasing our
understanding of the kinome and the nature of signaling networks.
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