Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A
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 <2.5 x 10/sup -4/ grains/..mu..m/sup 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 (/sup 125/I-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 /sup 125/I-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.
- Research Organization:
- Univ. of California, San Francisco, CA (United States)
- OSTI ID:
- 6844616
- Journal Information:
- J. Cell Biol.; (United States), Vol. 93:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL MEMBRANES
AUTORADIOGRAPHY
CONCANAVALIN
TISSUE DISTRIBUTION
ANIMAL CELLS
ELECTRON MICROSCOPY
ERYTHROCYTES
IODINE 125
MEASURING METHODS
MOLECULAR BIOLOGY
PROBES
QUANTITATIVE CHEMICAL ANALYSIS
AGGLUTININS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CHEMICAL ANALYSIS
DAYS LIVING RADIOISOTOPES
DISTRIBUTION
ELECTRON CAPTURE RADIOISOTOPES
HEMAGGLUTININS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
MATERIALS
MEMBRANES
MICROSCOPY
NUCLEI
ODD-EVEN NUCLEI
RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques