A novel method for measuring protein expression at the cell surface.
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)
Stoorvogel
W
W
Department of Cell Biology, University of Utrecht, Medical School, The Netherlands.
Oorschot
V
V
Neve
B
B
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
D001212:Asialoglycoproteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D017527:Receptor, IGF Type 2; D015100:3,3'-Diaminobenzidine; D000254:Sodium-Potassium-Exchanging ATPase