Differential labeling of cell-surface and internalized proteins after antibody feeding of live cultured neurons.
J Vis Exp
; (84): e51139, 2014 Feb 12.
Article
en En
| MEDLINE
| ID: mdl-24561550
ABSTRACT
In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking ("antibody feeding") to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.
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1
Banco de datos:
MEDLINE
Asunto principal:
Técnica del Anticuerpo Fluorescente
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Receptores de Superficie Celular
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Proteínas del Tejido Nervioso
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Neuronas
Límite:
Animals
Idioma:
En
Revista:
J Vis Exp
Año:
2014
Tipo del documento:
Article