Peeling as a novel, simple, and effective method for isolation of apical membrane from intact polarized epithelial cells.
Apical membrane of polarized epithelial cells is generally isolated by physicochemical methods, that is, precipitation with polyethylene glycol (PEG) or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation. However, these protocols are considerably sophisticated and frequently accompanied by impurities (e.g., contaminations of basolateral membrane and intracellular organelles), particularly by inexperienced investigators. We have developed a simple and effective method for isolation of apical membrane from intact polarized renal tubular epithelial cells. On the basis of hydrous affinity and/or ionic interaction, the apical membrane could be efficiently peeled from the cells by four different materials-Whatman filter paper, nitrocellulose membrane, cellophane, and glass coverslip-all of which are available in most research laboratories. Phase-contrast and laser-scanning confocal microscopic examinations using anti-ZO-1 antibody showed that other parts of the cells, particularly tight junction complex, remained intact after peeling by all four of these surfaces. Western blot analyses of gp135 (apical membrane marker) and of Na(+)/K(+)-ATPase, LAMP-2, COX-4, and calpain-1 (markers of basolateral membrane, lysosome, mitochondria, and cytosolic compartment, respectively) revealed that peeling with Whatman filter paper and glass coverslip was most and second-most effective, respectively, without any contaminations from basolateral membrane and other intracellular organelles that could be detected in the samples isolated by peeling with nitrocellulose membrane and cellophane and by conventional methods (i.e., precipitation with PEG or MgCl(2) followed by differential centrifugation or sucrose density gradient centrifugation). Our physical method is very simple, easy to follow (even by inexperienced investigators), time-saving, and cost-effective with a higher efficiency (as compared with conventional methods) for isolation of apical membrane from polarized epithelial cells.
Fong-ngern
Kedsarin
K
Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Chiangjong
Wararat
W
Thongboonkerd
Visith
V
D000818:Animals; D002448:Cell Adhesion; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D007668:Kidney / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013499:Surface Properties; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D062826:Zonula Occludens-1 Protein; C001643:podocalyxin