Rapid affinity purification of intracellular organelles using a twin strep tag

• Published in: Journal of cell science Vol. 132; no. 24
• Main Authors: Xiong, Jian, He, Jingquan, Xie, Wendy P, Hinojosa, Ezekiel, Ambati, Chandra Shekar R, Putluri, Nagireddy, Kim, Hyun-Eui, Zhu, Michael X, Du, Guangwei
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Ltd 13.12.2019
• Subjects: Chromatography, Affinity - methods; HeLa Cells; Humans; Lysosomes - metabolism; Mechanistic Target of Rapamycin Complex 1 - metabolism; Mitochondria - metabolism; Organelles - metabolism; Peroxisomes - metabolism; Tools and Resources; Twin strep tag; Lysosomes; Mitochondria; Peroxisomes
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.235390

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 the twin strep tag and streptavidin variants. With this method, we can isolate desired organelles with high purity and yield in 3 min from the post-nuclear supernatant of mammalian cells or less than 8 min 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.