Isolation of Rough and Smooth Membrane Domains of the Endoplasmic Reticulum from Rat Liver

Publisher Summary This chapter describes procedures for isolating rough and smooth membrane domains of the endoplasmic reticulum from rat liver. Blot the livers using lint-free tissues and transfer to a pre-weighed beaker containing ice-cold 0.25 M sucrose and weigh. Blot the livers using lint-free tissues and chop them finely with scissors. Allow the pieces to fall into the mortar of the tissue grinder. Using a fourth new syringe and needle, very gently insert needle to the bottom of the tube again and very carefully inject 1.4 ml of TM fraction to produce the bottom layer of the gradient. Turn tubes upside down on a lint-free tissue for approximately 30 s to allow residual supernatant to drip out. Wipe edges of the tube using a lint-free tissue wrapped around a glass rod. Once sequential rinsing is done, add sucrose-imidazole to tube so that the final volume of sucrose-imidazole added is equivalent to approximately 11 ml. Both microsomal fractions were shown previously to be enriched in enzyme markers for the endoplasmic reticulum and reduced in enzyme markers for Golgi and plasma membrane derivatives.