Method Article

Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative

July 8th, 2025

In This Article

Abstract

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Source: Tham, D. K. L., et al., Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation. J. Vis. Exp. (2017).

This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

Protocol

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation

NOTE: In the following, we describe a typical pulse-chase biotinylation experiment used in this instance to track the endocytosis of AQP4 in astrocytes. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see Table of Materials).

  1. Prepare cultures of mouse cortical ....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Bovine serum albuminSigma-AldrichA9647-50
Bromophenol blueBio-Rad#1610404
Complete protease inhibitor cocktailSigma-Aldrich11697498001
Disodium ethylenediaminetetraacetate dihydrate (EDTA)Bio-Rad#1610729
Dithiothreitol (DTT)Bio-Rad#1610611
Dulbecco's Modified Eagle Medium (DMEM)Gibco/Thermo Fisher Scientific11960-044
EZ-Link Sulfo-NHS-SS-BiotinThermo Fisher Scientific#21331
Fetal bovine serumGibco/Thermo Fisher Scientific16000-044
GlycerolFisher ScientificBP229-1
GlycineSigma-AldrichG8898
IodoacetamideBio-Rad#163-2109
L-glutamineGibco/Thermo Fisher Scientific25030-081
Penicillin/streptomycinGibco/Thermo Fisher Scientific15140-122
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)Jackson ImmunoResearch Laboratories111-035-045
Phosphate buffer salineGibco/Thermo Fisher Scientific10010-023
Reduced glutathioneSigma-AldrichG6529
Sodium chloride (NaCl)Fisher ScientificS271-500
Sodium dodecyl sulfate (SDS)Sigma-Aldrich862010
Sodium hydroxide (NaOH)Fisher ScientificS318-100
Streptavidin agarose resinThermo Fisher Scientific#20347
Rabbit polyclonal anti-AQP4 antibodyAlomoneAQP-004
Tris base (Trizma base)Fisher ScientificBP152-1
Tris-HClFisher ScientificBP153-1
Triton X-100Fisher ScientificBP151-500

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Tags

Protein InternalizationBiotinylation AssayCell Surface ProteinsStreptavidin PurificationWestern Blot AnalysisAstrocyte CulturesCleavable BiotinReducing Agent TreatmentStreptavidin AgaroseGel Electrophoresis

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