Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

• DOI: 10.3791/51087
• Publication Date: 2014-08-15T23:59:20Z
• Authors: Matthew Rames; Yadong Yu; Gang Ren
• Keywords: Cryo-Electron Microscopy; Negative stain; Structural Biology

Structural determination of proteins is rather challenging for with molecular masses between 40 - 200 kDa. Considering that more than half natural have a mass kDa1,2, robust and high-throughput method nanometer resolution capability needed. Negative staining (NS) electron microscopy (EM) an easy, rapid, qualitative approach which has frequently been used in research laboratories to examine protein structure protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially lipoproteins usually form presenting rouleaux artifacts. By using images from cryo-electron (cryo-EM) as standard, the key parameters specimen preparation conditions were recently screened reported optimized protocol (OpNS), modified 3 . Artifacts like can be greatly limited by OpNS, additionally providing high contrast along reasonably high‐resolution (near 1 nm) small asymmetric proteins. These high-resolution are even favorable individual (a single object, no average) 3D reconstruction, such 160 kDa antibody, through tomography4,5. Moreover, OpNS high‐throughput tool hundreds samples For example, previously published mechanism 53 cholesteryl ester transfer (CETP) involved screening imaging 6. cryo-EM rarely successfully less yet publish any study involving over one hundred sample conditions, it fair call studying Hopefully presented here useful push boundaries EM accelerate studies into structure, dynamics mechanisms.