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 membrane, Bicarbonate transporter, Micro-algae, Photosynthesis, Pyrenoid
Materials 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
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