Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin.
An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.
D001345:Autoradiography; D001426:Bacterial Proteins; D001710:Biotin; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D008856:Microscopy, Fluorescence; D019809:Streptavidin
D001426:Bacterial Proteins; D006023:Glycoproteins; D003208:Concanavalin A; D001710:Biotin; D019809:Streptavidin