title: Acceleratingin vitrostudies on circadian clock systems using an automated sampling device.
abstract: KaiC, a core protein of the cyanobacterial circadian clock, is rhythmically autophosphorylated and autodephosphorylated with a period of approximately 24 h in the presence of two other Kai proteins, KaiA and KaiB.In vitroexperiments to investigate the KaiC phosphorylation cycle consume considerable time and effort. To automate the fractionation, quantification, and evaluation steps, we developed a suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software. Eight sample tables can be controlled independently at different temperatures within a fluctuation of ±0.01°C, enabling investigation of the temperature dependency of clock activities simultaneously in a single experiment. The suite includes an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability. Multiple lanes on a gel can be detected quasi-automatically through an auto-detection procedure implemented in the software, with or without correction for lane 'smiling.' To demonstrate the performance of the suite, robustness of the period against temperature variations was evaluated using 32 datasets of the KaiC phosphorylation cycle. By using the software, the time required for the analysis was reduced by approximately 65% relative to the conventional method, with reasonable reproducibility and quality. The suite is potentially applicable to other clock or clock-related systems in higher organisms, relieving users from having to repeat multiple manual sampling and analytical steps.
authors.affiliation: Research Center of Integrative Molecular Systems, Institute for Molecular Science, NINS, Aichi 444-8585, Japan; Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Aichi 444-8585, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Furuike
authors.affiliation: Research Center of Integrative Molecular Systems, Institute for Molecular Science, NINS, Aichi 444-8585, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Abe
authors.affiliation: Research Center of Integrative Molecular Systems, Institute for Molecular Science, NINS, Aichi 444-8585, Japan; Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Aichi 444-8585, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Mukaiyama
authors.affiliation: Research Center of Integrative Molecular Systems, Institute for Molecular Science, NINS, Aichi 444-8585, Japan; Department of Functional Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Aichi 444-8585, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Akiyama
keywords: circadian clock; cyanobacteria; kai; rhythm; temperature compensation