Rapid affinity purification of intracellular organelles using twin strep tag

Abstract

Cells are internally organized into compartmentalized organelles that execute specialized functions. To understand the functions of individual organelles and their regulations, it is critical to resolve the compositions of individual organelles, which relies on a rapid and efficient isolation method for specific organellar populations. Here, we introduce a robust affinity purification method for rapid isolation of intracellular organelles, e.g., lysosomes, mitochondria and peroxisomes, by taking advantage of the extraordinarily high affinity between twin strep tag and streptavidin variants. With this method, we can isolate desired organelles with high purity and yield in 3 minutes from the post-nuclear supernatant of mammalian cells or less than 8 minutes for the whole purification process. Using lysosomes as an example, we show that the rapid procedure is especially useful for studying transient and fast cellular activities, such as organelle-initiated signaling and organellar contents of small molecular metabolites. Therefore, our method offers a powerful tool to dissect spatiotemporal regulation and functions of intracellular organelles.