5] Isolation of microsomal subfractions by use of density gradients

Abstract

This chapter provides information on the isolation of microsomal subfractions by use of density gradients. To avoid contamination of microsomes by fragments of organelles other than the endoplasmic reticulum (ER), relatively gentle homogenization (4 x 400 rpm) is required. Isolation of rough and smooth microsomes with a sucrose gradient containing cations is an effective and rapid procedure, but both cation-free water and careful maintenance of laboratory animals are required to avoid aggregation. It is possible to carry out density gradient centrifugation using metrizamide gradients because this substance has a low viscosity in aqueous solutions even when gradients with high density are formed. Metrizamide is nonionic, does not inhibit microsomal enzymes, and has no detergent effects on membranes, making this solute advantageous for density gradient centrifugation of microsomal fractions. It appears that deoxycholate liberates membrane fragments, which upon gradient centrifugation equilibrate in different bands. The presence of similar fragments in high concentration within the same band may result in aggregation of these fragments to larger particles.