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Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin
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SYSNO ASEP 0582456 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin Author(s) Masuda, Takako (MBU-M) ORCID
Majerová, Dominika (MBU-M)
Piwosz, Kasia (MBU-M) ORCID
Tsurumaki, Tatsuhiro (MBU-M)
Fujita, Y. (JP)
Prášil, Ondřej (MBU-M) RID, ORCIDArticle number e65168 Source Title Jove-Journal of Visualized Experiments - ISSN 1940-087X
Roč. 2023, č. 199 (2023)Number of pages 16 s. Language eng - English Country US - United States Keywords Chlorophyl ; phycoerythrin ; cyanobacterium ; phycourobilin OECD category Microbiology R&D Projects GA20-17627S GA ČR - Czech Science Foundation (CSF) Method of publishing Limited access Institutional support MBU-M - RVO:61388971 UT WOS 001159858200019 EID SCOPUS 85172771682 DOI 10.3791/65168 Annotation Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin-and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells. © 2023 JoVE Journal of Visualized Experiments. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2024 Electronic address https://www.jove.com/t/65168/immunocytochemical-visualization-proteins-from-cyanobacterial-cells
Number of the records: 1