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Open AccessProtocol

Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

1
Chemistry & Chemical Biology, University of California, Merced, CA 95343, USA
2
Center for Cellular and Biomolecular Machines, University of California, Merced, CA 95343, USA
3
School of Natural Sciences, University of California, Merced, CA 95343, USA
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Center for Circadian Biology, University of California, San Diego, La Jolla, CA 92093, USA
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Quantitative & Systems Biology, University of California, Merced, CA 95343, USA
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Health Sciences Research Institute, University of California, Merced, CA 95343, USA
*
Author to whom correspondence should be addressed.
Current address: Biochemistry & Molecular Biology, Monash University, 3800 Melbourne, Australia.
Methods Protoc. 2019, 2(2), 42; https://doi.org/10.3390/mps2020042
Received: 21 April 2019 / Revised: 20 May 2019 / Accepted: 22 May 2019 / Published: 24 May 2019
Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks. View Full-Text
Keywords: fluorescence; circadian clock; cyanobacteria; protein; phosphorylation fluorescence; circadian clock; cyanobacteria; protein; phosphorylation
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MDPI and ACS Style

Heisler, J.; Chavan, A.; Chang, Y.-G.; LiWang, A. Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock. Methods Protoc. 2019, 2, 42.

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