An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM.

ABSTRACT: In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from ce

SUBMITTER: Zhao Q 

PROVIDER: S-EPMC11164986 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature