Nature research paper: Structure of the lysosomal mTORC1–TFEB–Rag–Ragulator megacomplex
. In brief, Sf9 cells were pelleted after 72 h of baculovirus infection and lysed in the wash buffer with 1% Triton X-100 and protease inhibitor. The cleared supernatant after centrifugation was applied to Ni-nitrilotriacetic acid gravity column , washed with the wash buffer containing 200 mM NaCl, and eluted with the wash buffer with 250 mM imidazole. The elution was then applied to glutathione Sepharose 4B gravity column, washed with the wash buffer.
. On the other hand, Strep-Tactin resin was used for the affinity purification and the complex was eluted with the wash buffer containing 10 mM-desthiobiotin. The elution was diluted into equal volume of salt-free buffer and applied to a 5 ml HiTrap Q column . The mTORC1 complex and free RAPTOR were separated by a 100 ml salt gradient with salt-free buffer and high salt buffer . The fractions containing mTORC1 complex and free Raptor were concentrated to 1.3 and 0.
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