Fig. 5: Purification and characterization of 20S proteasome using SNAP-MS.

a Schematic representation of hierarchy, purification, and rearrangement of 20S proteasome. b Native PAGE, SEC, and native stain EM characterization of purified 20S, with α and β subunits expressed from separate plasmids. The SDS-PAGE experiment was independently repeated three times, yielding consistent results. c Detection of β subunit from samples containing 20S expressed using separate plasmids. Left: SDS-PAGE of products from the purification of Twin-Strep- or His-tagged proteins using N₃- or ST-modified beads (l: cell lysate; p: pure 20S; b: blank). Right: Western blot (with anti-Strep Ab) of the 20S purified using SNAP beads synthesized with different concentrations of Linker 2, showing the presence of β subunit. d Native mass spectra of purified 20S expressed from separate plasmids. e Native mass spectra of purified 20S expressed from a single plasmid. f 3D-reconstructed density map of the purified intact 20S structure at a resolution of 3.16 Å using 120 micrographs. Source data are provided as a Source Data file.