The Journal of Cell Biology, Vol 92, 44-52, Copyright © 1982 by The Rockefeller University Press
Spectroscopic assays for measuring quantities of erythrocyte membrane "halves"
KA Fisher
The quantities of outer and inner"halves" produced by freeze-fracturing
human erythrocyte membranes have been measured by visible and fluorescence
spectroscopy. Assays have been developed that are based on the use of two
membrane surface markers: hemoglobin (Hb), a native marker for the
cytoplasmic side of the membrane, and fluoresceinated concanavalin A
(FITC-Con-A), a marker for the extracellular side. Hb absorbance is
proportional to the fraction of cytoplasmic "half" membranes, and FITC
fluorescence is proportional to the fraction of extracellular "halves." A
procedure is described for the preparation of surface-labeled, intact
erythrocytes suitable for the formation of homogeneous, planar cell
monolayers of square-centimeter dimensions on polylysine-treated glass
(PL-glass). Cell monolayers were frozen and fractured, and the fractions of
absorbance and fluorescence in each of the two split portions determined.
The PL-glass portion of membrane contained a substantially higher ratio of
fluorescence to absorbance than unsplit controls, and its paired portion, a
complementary lower ratio, demonstrating that the PL-glass portion was
significantly enriched in extracellular "half" membrane. Experiments
investigating split membrane recovery show that the double labeled membrane
splitting technique is well suited to analysis of the transmembrane
distribution of membrane lipids and polypeptides using methods that do not
require quantitation by electron microscopy.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
