Cell Surface Protein Biotinylation and Imaging

We performed a tracer experiment using a primary amine-reactive biotinylation reagent which labels proteins and primary amine-containing macromolecules on a cell surface [23 (link)]. Epithelia grown on 12-mm filters were incubated in solution A with 0.01mg/ml EZ-Link Sulfo-NHS-Biotin (Life Technologies) in the basal side for 10 min at 37°C. Then epithelia were fixed with 10% paraformaldehyde for 7 min and pemeabilized with Triton X-100. The bound biotin was detected by alexa fluor 555 streptavidin (S-32355; Life Technologies). The samples were imaged on a Zeiss LSM700 confocal microscope in a similar manner as described in Immunocytochemistry.

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Tokuda S., Kim Y.H., Matsumoto H., Muro S., Hirai T., Mishima M, & Furuse M. (2015). Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia. PLoS ONE, 10(12), e0145522.

Publication 2015

EZ-Link Sulfo-NHS-Biotin alexa fluor 555 streptavidin LSM700 confocal microscope

Alexa fluor 555 Amine Biotin Biotinylation Cell Confocal microscope Epithelia Immunocytochemistry Paraformaldehyde Proteins Streptavidin Sulfo nhs biotin Triton x 100

Corresponding Organization : National Institute for Physiological Sciences

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Variable analysis

independent variables

Incubation of epithelia in solution A with 0.01 mg/ml EZ-Link Sulfo-NHS-Biotin in the basal side for 10 min at 37°C

dependent variables

Labeling of proteins and primary amine-containing macromolecules on the cell surface with the biotinylation reagent

control variables

Epithelial cells grown on 12-mm filters
Fixation of epithelia with 10% paraformaldehyde for 7 min
Permeabilization of epithelia with Triton X-100
Detection of bound biotin with Alexa Fluor 555 streptavidin

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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