دورية أكاديمية
Isolation of the Pistil-Stimulated Pollen Tube Secretome.
| العنوان: | Isolation of the Pistil-Stimulated Pollen Tube Secretome. |
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| المؤلفون: | Hafidh S; Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic. hafidh@ueb.cas.cz., Honys D; Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic. david@ueb.cas.cz. |
| المصدر: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2020; Vol. 2160, pp. 41-72. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press, |
| مواضيع طبية MeSH: | Exocytosis*, Pollen Tube/*metabolism , Proteome/*metabolism , Proteomics/*methods, Chromatography, Liquid/methods ; Magnoliopsida ; Mass Spectrometry/methods ; Ovule/metabolism ; Plant Proteins/chemistry ; Plant Proteins/metabolism ; Proteome/chemistry |
| مستخلص: | Detection of secreted proteins and peptides during pollen tube guidance has been impeded due to lack of techniques to capture the pollen tube secretome without contamination from the female secreted proteins. Here we present a protocol to detect tobacco pollen tube secreted proteins, semi-in vivo pollen tube secretome assay (SIV-PS), following pollen tube crosstalk with the female reproductive tissues. This method combines the advantages of in vivo pollen tube-pistil interaction and filter-aided sample preparation (FASP) techniques to obtain an in-depth proteome coverage. The SIV-PS method is rapid, efficient, inexpensive, does not require specialized equipment or expertise, and provides a snapshot of the ongoing molecular interplay. We show that the secretome obtained is of greater purity (<1.4% ADH activities) and that pollen tubes are physiologically and cytologically unaffected. A compendium of quality controls is described and a rough guide on downstream bioinformatics analysis is outlined. The SIV-PS method is applicable to all studies of protein secretion using pollen tube as a model and can be easily adapted to other flowering species with modification. The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis. |
| فهرسة مساهمة: | Keywords: Cell–cell communication; Glycosylation; LC-MS/MS; Pollen tube guidance; Protein secretion; Sexual plant reproduction |
| المشرفين على المادة: | 0 (Plant Proteins) 0 (Proteome) |
| تواريخ الأحداث: | Date Created: 20200613 Date Completed: 20210311 Latest Revision: 20210311 |
| رمز التحديث: | 20240829 |
| DOI: | 10.1007/978-1-0716-0672-8_4 |
| PMID: | 32529428 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1940-6029 |
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| DOI: | 10.1007/978-1-0716-0672-8_4 |