Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label.
A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.
Busch
G
G
Institut für Klinische Chemie und Biochemie, Freie Universität, Berlin-West/Federal Republic of Germany.
Hoder
D
D
Reutter
W
W
Tauber
R
R
D000478:Alkylation; D000818:Animals; D001692:Biological Transport; D001710:Biotin; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006651:Histocytochemistry; D006801:Humans; D008099:Liver / Q000166:cytology; D008297:Male; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D001710:Biotin