A novel method for measuring protein expression at the cell surface

W Stoorvogel, V Oorschot, B Neve

    Research output: Contribution to journalArticleAcademicpeer-review


    All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3'-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

    Original languageEnglish
    Pages (from-to)1201-9
    Number of pages9
    JournalJournal of Cell Science
    Volume106 ( Pt 4)
    Publication statusPublished - Dec 1993


    • 3,3'-Diaminobenzidine
    • Animals
    • Asialoglycoproteins
    • Biological Transport
    • Cell Line
    • Cell Membrane
    • Cross-Linking Reagents
    • Endocytosis
    • Humans
    • Membrane Proteins
    • Receptor, IGF Type 2
    • Sodium-Potassium-Exchanging ATPase


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