Cryohomogenization of liver to make in vitro preparations of rough endoplasmic reticulum and other organelles

Abstract

Cell fractionation has been used for many years to isolate organelles for biochemical study. Typically, fresh tissue is homogenized in a suitable buffer, and the various size-classes of organelles are then isolated by differential centrifugation. One of the common fractions is the microsomal fraction, which consists largely of vesiculated rough and smooth endoplasmic reticulum, and of Golgi elements.In the past we have investigated bound polysomes on the rough endoplasmic reticulum (RER) by electron microscopy, and have used cell fractionation and EM to study the orientation of ribosomes in polysomes bound to rough microsomal vesicles. However, the value of the cell fractionation material in our work has been limited by the small size of the microsomal vesicles, which are produced by vesicular fragmentation of the endoplasmic reticulum during tissue homogenization. For our work we need in vitro preparations in which the RER retains its cisternal form, rather than being fragmented into small vesicles. This report describes a method we have devised that can yield relatively intact RER in vitro.