Rapid affinity purification of intracellular organelles using a twin strep tag
| Autor: | Michael X. Zhu, Guangwei Du, Hyun-Eui Kim, Ezekiel Hinojosa, Chandrashekar R. Ambati, Jingquan He, Nagireddy Putluri, Jian Xiong, Wendy P. Xie |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Streptavidin
Strep-tag Mitochondrion Biology Mechanistic Target of Rapamycin Complex 1 Chromatography Affinity 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Affinity chromatography Organelle Peroxisomes Humans 030304 developmental biology Organelles 0303 health sciences Cell Biology Peroxisome Cell biology Tools and Resources Mitochondria chemistry Lysosomes 030217 neurology & neurosurgery Intracellular organelles HeLa Cells |
| Zdroj: | J Cell Sci |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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