title: AN OPTIMIZED METHOD FOR THE ISOLATION OF NUCLEI FROM CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)(1).
abstract: The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches.
authors.affiliation: University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Silesia, Department of Genetics, Jagiellonska 28, 40-032, Katowice, PolandMax-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Flavia Vischi
authors.initials: FV
authors.lastname: Winck
authors.affiliation: University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Silesia, Department of Genetics, Jagiellonska 28, 40-032, Katowice, PolandMax-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Kwasniewski
authors.affiliation: University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Silesia, Department of Genetics, Jagiellonska 28, 40-032, Katowice, PolandMax-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wienkoop
authors.affiliation: University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Silesia, Department of Genetics, Jagiellonska 28, 40-032, Katowice, PolandMax-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany University of Potsdam, GoFORSYS Research Unit for Systems Biology, Institute of Biochemistry and Biology, Karl-Liebknecht-Straße 24-25, Haus 20, 14476 Potsdam-Golm, Germany Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Mueller-Roeber
keywords: 2D gel electrophoresis; Chlamydomonas; algae; nuclear proteins; nucleus; proteomics