Abstract
The method of immunoelectron microscopy is intended for localization of proteins inside the cells of Chlamydomonas reinhardtii or other microalgae and cyanobacteria. This protocol was used to study localization of carbonic anhydrase Cah3 with antibodies raised in rabbit, though it can be used to localize any other abundant protein. Primary rabbit antibodies are recommended because they react quickly and specifically with proteins of C. reinhardtii. If primary antibodies other than rabbit are used, the blocking procedure and time of incubation with primary and secondary antibodies should be adjusted.
Keywords: Ultrastructure, Chlamydomonas reinhardtii, Carbonic anhydrase, Localization
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Sinetova et al. (2012). We appreciate Prof. J. Moroney (Louisiana State University, USA) for providing the strain of C. reinhardtii 137mt+ and CCM-mutant cia3, Prof. G. Samuelsson (Umea University, Sweden) who kindly supplied us with primary antibodies against recombinant Cah3 and Cah3-MBP fusion protein. We thank Prof. Pronina N.A. and Dr. Kupriyanova E.V. for the physiological and molecular biological base of this protocol. This work was supported by a grant from the Russian Foundation for Basic Research (no. 10-04-01463), a grant from the Presidium of Russian Academy of Sciences (program “Molecular and Cell Biology”), and a grant from the Russian Ministry of Education and Science (no. 16.740.11.0176).
References
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