Electron Microscopy of Bound Polysomes on in Vitro Rough Endoplasmic Reticulum Prepared by Cryohomogenization

Abstract

We have previously described a cryohomogenization method for making in vitropreparations of rough endoplasmic reticulum (RER) and other organelles. The goal of that approach was to minimize the extensive vesicular fragmentation of the endoplasmic reticulum that occurs during homogenization for conventional cell fractionation. The microsome fraction of cell fractionation consists primarily of small vesicles derived from rough and smooth endoplasmic reticulum, and has been of great value in biochemical studies of protein synthesis and secretion. However, the small size of the microsome vesicles has made them less useful for in vitro studies of bound polysomes by electron microscopy. As a further development of our cryohomogenization approach, we here describe a method for removing larger particles and debris from the cryohomogenate by filtration, and the application of in vitro RER and other organelles to EM grid membranes for negative staining and viewing by electron microscopy.