Nature Methods2, 277 - 284 (2005)
Published online: 23 March 2005; | doi:10.1038/nmeth747
A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates
Melissa D Shults1, Kevin A Janes2, Douglas A Lauffenburger2, 3
& Barbara Imperiali1, 3
1
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
2
Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
3
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.
Correspondence should be addressed to Barbara Imperiali imper@mit.edu
New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase−associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.
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