title: A novel method for measuring protein expression at the cell surface.
abstract: 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)
authors.affiliation: Department of Cell Biology, University of Utrecht, Medical School, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Stoorvogel
authors.forename: V
authors.initials: V
authors.lastname: Oorschot
authors.forename: B
authors.initials: B
authors.lastname: Neve
mesh_terms: D015100:3,3'-Diaminobenzidine; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D004705:Endocytosis; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D001212:Asialoglycoproteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D015100:3,3'-Diaminobenzidine; D000254:Sodium-Potassium-Exchanging ATPase