Ultrastructural localization of de novo synthesized phosphatidylcholine in yeast cells by freeze-fracture electron microscopy.
STAR Protoc
; 2(4): 100990, 2021 12 17.
Article
en En
| MEDLINE
| ID: mdl-34934959
ABSTRACT
Phosphatidylcholine (PtdCho) is a major membrane phospholipid synthesized in the endoplasmic reticulum. Here, we provide a protocol using electron microscopy to localize PtdCho that is newly synthesized by the Kennedy pathway in yeast cells. The protocol consists of the administration of a clickable alkyne-containing choline analog to cells, quick-freezing, freeze-fracture replica preparation, conjugation of biotin-azide by click chemical reaction, and immunogold labeling. This protocol can be used to determine quantitatively to which membrane leaflets newly synthesized PtdCho is incorporated. For complete details on the use and execution of this protocol, please refer to Orii et al. (2021).
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fosfatidilcolinas
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Saccharomyces cerevisiae
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Microscopía Electrónica
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Técnica de Fractura por Congelación
Idioma:
En
Revista:
STAR Protoc
Año:
2021
Tipo del documento:
Article
País de afiliación:
Japón