Slides6 11.45.23 PM - MIT OpenCourseWare http:/ocw.mit.edu...

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MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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A B Which is a kinase domain? A, B, or both?
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Detecting Kinase Activity in vitro and in vivo I. In vitro activity assays (for purified kinases) A. 32 P incorporation assay B. Gel analysis with a phospho-specific antibody C. Kinase coupled-activity assay (lab sessions 13 and 14) II. Fluorescent peptide-based probes A. Environment-sensitive fluorescent probes B. Chelation-enhanced fluorescent probes III. In-vivo protein probes (FRET) IV. Laboratory report guidelines
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Detecting protein kinase activity in vitro and in vivo substrate O - O P NH2 NH2 OH O O - N N N substrate N N N + O O O N kinase + O O N - O P O P O - O P O P O P O O O H H H H O - O - O - O - O - H H H H OH H OH H ATP ADP Why are scientists interested in detecting kinase activity? Enables monitoring of kinase activity and inhibition Used for screening of drug candidates Facilitates the unraveling of complex cell-signalling pathways implicated in processes ranging from cell cycle regulation to cellular migration (including tumor metastasis).
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Detecting protein kinase activity in vitro and in vivo substrate O - O P NH2 NH2 OH O O - N N N substrate N N N + O O O N kinase + O O N - O P O P O - O P O P O P O O O H H H H O - O - O - O - O - H H H H OH H OH H ATP ADP Enzyme activity assays detect either product formation or starting material consumption.
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Detecting protein kinase activity in vitro and in vivo substrate O - O P NH2 NH2 OH O O - N N N substrate N N N + O O O N kinase + O O N - O P O P O - O P O P O P O O O H H H H O - O - O - O - O - H H H H OH H OH H ATP ADP Enzyme activity assays detect either product formation or starting material consumption. For kinases this means Detection of phosphorylated substrate (product) or Detection of ADP (product) formation or ATP (starting material) consumption
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Detecting protein kinase activity in vitro and in vivo substrate O - O P NH2 NH2 OH O O - N N N substrate N N N + O O O N kinase + O O N - O P O P O - O P O P O P O O O H H H H O - O - O - O - O - H H H H OH H OH H ATP ADP However, phosphorylation is spectroscopically silent . Neither the formation of phosphorylated product nor the conversion of ATP to ADP can be monitored directly using spectroscopy. The products appear identical to the starting materials due to identical absorption features and extinction coefficients.
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Kinase assays: in buffer, in cell lysate, in vivo Depending on your application, you should consider whether you need an assay that can be performed only with purified enzyme, in a cell lysate mixture, or in vivo. Some other considerations may include a protocol that is: Easy and safe to use General versus specific Continuous (versus an end-point assay) A turn-on versus a turn-off assay (some researchers prefer the generation of a positive signal rather than a decrease in signal) Amenable to high-throughput applications Sensitive to low levels of kinase activity
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This note was uploaded on 12/16/2011 for the course BIOLOGY 101 taught by Professor Mr.wallace during the Fall '11 term at Montgomery College.

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Slides6 11.45.23 PM - MIT OpenCourseWare http:/ocw.mit.edu...

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