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Title

In situ/Subcellular localization of arabinogalactan protein expression by fluorescent in situ hybridization, FISH

Authorsda Costa, Mário Luís; Solís, María Teresa ; Testillano, P. S. ; Coimbra, Silvia
KeywordsArabidopsis
Arabinogalactan proteins
FISH
Immunolocalization
RNA-Probes
Whole-mount
Issue Date3-Jul-2020
PublisherHumana Press
CitationThe Plant Cell Wall. Popper Z. (eds) Methods in Molecular Biology, vol 2149 (2020)
AbstractThe arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.
Description31 p. Methods Mol Biol 2149:403-427
Publisher version (URL)https://doi.org/10.1007/978-1-0716-0621-6_23
URIhttp://hdl.handle.net/10261/216692
DOIhttp://dx.doi.org/10.1007/978-1-0716-0621-6_23
ISBN978-1-0716-0619-3
Appears in Collections:(CIB) Libros y partes de libros
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