Journal article
In vitro protein kinase activity measurement by flow cytometry
Analytical biochemistry, Vol.383(2), pp.180-185
2008
DOI: 10.1016/j.ab.2008.08.026
PMID: 18796290
Abstract
Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.
Details
- Title: Subtitle
- In vitro protein kinase activity measurement by flow cytometry
- Creators
- Donald J Bernsteel - Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USADavid L Roman - Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USARichard R Neubig - Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USA
- Resource Type
- Journal article
- Publication Details
- Analytical biochemistry, Vol.383(2), pp.180-185
- Publisher
- Elsevier Inc
- DOI
- 10.1016/j.ab.2008.08.026
- PMID
- 18796290
- ISSN
- 0003-2697
- eISSN
- 1096-0309
- Language
- English
- Date published
- 2008
- Academic Unit
- Pharmacy; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984065382902771
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