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

Commun Biol. 2024 Jun 10;7(1):715. doi: 10.1038/s42003-024-06406-z.

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 cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).

MeSH terms

  • Antibodies, Immobilized* / chemistry
  • Antibodies, Immobilized* / immunology
  • Cryoelectron Microscopy* / methods
  • Graphite / chemistry
  • Humans

Substances

  • Antibodies, Immobilized
  • Graphite
  • graphene oxide