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This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for fractionation of other types of tissue. The protocol has two major steps 1) preparation of homogenate from dissected tissue and 2) separation of organelles by centrifugation of homogenates using a continuous sucrose gradient.