The Journal of Cell Biology, Vol 93, 155-163, Copyright © 1982 by The Rockefeller University Press
Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A
KA Fisher
The technique of monolayer freeze-fracture autoradiography (MONOFARG) has
been developed and the principles, quantitation, and application of the
method are described. Cell monolayers attached to polylysine- treated glass
were freeze-fractured, shadowed, and coated with dry, Parlodion-supported
Ilford L4 photographic emulsion at room temperature. Quantitative aspects
of MONOFARG were examined using radioiodinated test systems. Background was
routinely less than 2.5 X 10(-4) grains/microns 2/day, the highest overall
efficiency was between 25% and 45%, and grain density and efficiency were
dependent on radiation dose for iodine-125 and D-19 development. Corrected
grain densities were linearly proportional to iodine-125 concentration. The
method was applied to an examination of the transmembrane distribution of
radioiodinated and fluoresceinated concanavalin A (125I-FITC-Con-A). Human
erythrocytes were labeled, column-purified, freeze-dried or
freeze-fractured, autoradiographed, and examined by electron microscopy.
The number of silver grains per square micrometer of unsplit single
membrane was essentially identical to that of split extracellular membrane
"halves." These data demonstrate that 125I-FITC- Con-A partitions
exclusively with the extracellular "half" of the membrane upon
freeze-fracturing and can be used as a quantitative marker for the fraction
of extracellular split membrane "halves." This method should be able to
provide new information about certain transmembrane properties of
biological membrane molecules and probes, as well as about the process of
freeze-fracture per se.
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