This chapter describes the preparation and isolation of highly purified endoplasmic reticulum (ER) from the endosperm of developing and germinating castor bean (Ricinus communis) seeds to provide a purified organelle fraction for differential proteomic analyses. The method uses a two-step ultracentrifugation protocol first described by Coughlan (1) and uses sucrose density gradients and a sucrose flotation step to yield purified ER devoid of other contaminating endomembrane material. Using a combination of one dimensional (1D) and two dimensional (2D) gel electrophoresis the complexity and reproducibility of the protein profile of the purified organelle is evaluated prior to detailed proteomic analyses using mass spectrometry based techniques.