Abstract
Stochastic diffusion of a solution of fluorophores after photoselection reduces the polarization of emission, or fluorescence anisotropy. Because this randomization process is slower for larger molecules, fluorescence anisotropy is effective for measuring the kinetics of protein-binding events. Here, we describe how to use the technique to carry out real-time observations in vitro of the cyanobacterial circadian clock.
Highlights
Organisms from all domains of life display circadian (~24 h) rhythms in their metabolism, physiology, and behavior that arose as an adaptation to daily cycles of ambient light and temperature [1].These endogenous rhythms are generated by intracellular circadian clocks
The cyanobacterial clock offers a unique opportunity in this regard
Fluorescence anisotropy is widely used by biochemists
Summary
Organisms from all domains of life display circadian (~24 h) rhythms in their metabolism, physiology, and behavior that arose as an adaptation to daily cycles of ambient light and temperature [1]. Time points of in vitro cyanobacterial clock reactions are analyzed ex post facto using denaturing polyacrylamide gel electrophoresis (SDS PAGE) to resolve different states of KaiC phosphorylation. We demonstrate that in vitro fluorescence spectroscopy can directly monitor circadian rhythms of protein-protein interactions in the cyanobacterial clock in real time by utilizing clock rhythms of protein-protein interactions in the cyanobacterial clock in real time by utilizing clock proteins labeled with fluorescent dyes. 6-iodoacetamidofluroescein (6IAF) labeled KaiB allows direct observations of real-time population shifts between free KaiB (daytime) and bound KaiB (nighttime) (Figure 1). This fluorescence method shifts between free KaiB (daytime) and bound KaiB (nighttime) (Figure 1).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
