Using Percoll Gradient Fractionation to Study the Endocytic Trafficking of the EGFR

Scientists have separated subcellular compartments based on differences in their densities by gradient centrifugation for decades. With the proper equipment and thoughtful experimental design, density gradients are a reliable method for enriching various intracellular compartments to assess their protein composition, morphology, or biochemical activity. While variations of this basic technique have been used for separating whole cells, endosomes, synaptic vesicles, and viruses, we have found it especially useful for resolving the compartments of the endocytic pathway. In particular, this technique has been valuable for studying the regulation and signaling of the Epidermal Growth Factor Receptor (EGFR) while undergoing endocytic trafficking. In this article we detail the technical aspects of utilizing Percoll gradients to study the endocytic trafficking of the EGFR and associated proteins.