AJP - Heart and Circulatory Physiology, Vol 241, Issue 6 891-H893, Copyright © 1981 by American Physiological Society
A technique for freeze fracturing minute amounts of isolated cardiac membrane
Y. Shibata and C. K. Manjunath
Electron microscopy (EM) of freeze-fractured membranes provides more
information about internal membrane structure than EM of thin-sectioned or
negatively stained material. However, it has heretofore been impractical to
use freeze fracture routinely for analysis of highly purified membrane
fractions obtainable in small (micrograms) amounts, because the technique,
when conventionally applied to minute pellets, yields only one fracture of
unpredictable quality; it also precludes in parallel biochemical studies by
using up the entire preparation. To solve this problem, we have developed a
method for freeze fracturing tiny droplets of suspended membranes
containing 1-10 micrograms membrane protein, thereby allowing both multiple
fractures and biochemical studies. Before fracture, the final membrane
fractions can be concentrated, subjected to experimental manipulations,
cross-linked, and glycerinated in a dialysis bag. The technique is
illustrated on isolated gap junctions from rabbit hearts, which were chosen
because their unique internal membrane structure allows unequivocal
identification of membrane type based on structural criteria.
