A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates - PubMed (original) (raw)

doi: 10.1038/nmeth747. Epub 2005 Mar 23.

Affiliations

PMID: 15782220
DOI: 10.1038/nmeth747

A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

Melissa D Shults et al. Nat Methods. 2005 Apr.

Abstract

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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Comment in

Measuring kinase activity: finding needles in a haystack.
Turk BE. Turk BE. Nat Methods. 2005 Apr;2(4):251-2. doi: 10.1038/nmeth0405-251. Nat Methods. 2005. PMID: 15782214 No abstract available.

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A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates - PubMed (original) (raw)