ISOLATION OF PLASMA-MEMBRANE FRACTIONS FROM THE INTESTINAL EPITHELIALMODEL-T84

The human intestinal epithelial cell line T84 is widely used as a mode l for studies of Cl- secretion and crypt cell biology. We report a fra ctionation approach that permits separation of purified apical and bas olateral T84 plasma membrane domains. T84 cellular membranes were isol ated by nitrogen cavitation and differential centrifugation from monol ayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alk aline phosphatase) and basolateral (Na+-K+-ATPase) plasma membrane mar kers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. B uffer conditions were defined that permitted separation of enriched ap ical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical a nd basolateral surface labeling studies using trace iodinated wheat ge rm agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.