Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons
Nissa L Carrodus, Kathleen Sue-Lyn Teng, Kathryn M Munro, Matthew J Kennedy, Jenny M Gunnersen
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | JOURNAL OF VISUALIZED EXPERIMENTS | Published : 2014
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 surfa..View full abstract
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Awarded by National Health and Medical Research Council, Australia
Awarded by NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
The authors thank Teele Palumaa for assistance with the figures. Funded by Project Grant 1008046 from the National Health and Medical Research Council, Australia.