An optimized method for the isolation of nuclei from chlamydomas Reinhardtii (Chlorophyceae)
- 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. TheThe 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.…
Author details: | Flavia Vischi Winck, Miroslaw Kwasniewski, Stefanie Wienkoop, Bernd Müller-RöberORCiDGND |
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DOI: | https://doi.org/10.1111/j.1529-8817.2011.00967.x |
ISSN: | 0022-3646 |
Title of parent work (English): | Journal of phycology |
Publisher: | Wiley-Blackwell |
Place of publishing: | Malden |
Publication type: | Article |
Language: | English |
Year of first publication: | 2011 |
Publication year: | 2011 |
Release date: | 2017/03/26 |
Tag: | 2D gel electrophoresis; Chlamydomonas; algae; nuclear proteins; nucleus; proteomics |
Volume: | 47 |
Issue: | 2 |
Number of pages: | 8 |
First page: | 333 |
Last Page: | 340 |
Funding institution: | BMBF [FKZ 0313924]; DAAD [A/06/04209]; Fonds der Chemischen Industrie [0164389] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
Peer review: | Referiert |