Published: Vol 6, Iss 13, Jul 5, 2016 DOI: 10.21769/BioProtoc.1864 Views: 12533
Reviewed by: Maria SinetovaCindy AstAnonymous reviewer(s)
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Abstract
Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO2-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et al., 2015), and LCIA (chloroplast envelope anion channel; Yamano et al., 2015) in Chlamydomonas reinhardtii. The protocol described here could be useful for observing the protein of interest in other algae cells.
Keywords: Chloroplast membraneMaterials and Reagents
Equipment
Note: these were the instruments used throughout our experiments, but any company is OK.
Procedure
Notes
We highly recommend that user also performs the same experiment using a target gene mutant as a negative control to distinguish true signals from artifact.
Recipes
Acknowledgments
We thank Lianyong Wang for technical assistance. This work was supported by the Japan Society for the Promotion of Science KAKENHI Grants 25120714 (to H.F.) and 25840109 (to T.Y.) and the Japan Science and Technology Agency Advanced Low Carbon Technology Research and Development Program.
References
Article Information
Copyright
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Yamano, T. and Fukuzawa, H. (2016). Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii. Bio-protocol 6(13): e1864. DOI: 10.21769/BioProtoc.1864.
Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Immunodetection
Cell Biology > Cell imaging > Fluorescence
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