Electrophoretic exclusion microscale sample preparation for cryo-EM structural determination of proteins

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2 Scopus citations

Abstract

Transmission electron microscopy (TEM) of biological samples has a long history and has provided many important insights into fundamental processes and diseases. While great strides have been made in EM data collection and data processing, sample preparation is still performed using decades-old techniques. Those sample preparation methods rely on extensive macroscale purification and concentration to achieve homogeneity suitable for high-resolution analyses. Noting that relatively few bioparticles are needed to generate high-quality protein structures, this work uses microfluidics that can accurately and precisely manipulate and deliver bioparticles to grids for imaging. The use of microfluidics enables isolation, purification, and concentration of specific target proteins at these small scales and does so in a relatively short period of time (minutes). These capabilities enable imaging of more dilute solutions and obtaining pure protein images from mixtures. In this system, spatially isolated, purified, and concentrated proteins are transferred directly onto electron microscopy grids for imaging. The processing enables imaging of more dilute solutions, as low as 5 × 10-6 g/ml, with small total amounts of protein (<400 pg, 900 amol). These levels may be achieved with mixtures and, as proof-of-principle, imaging of one protein from a mixture of two proteins is demonstrated.

Original languageEnglish (US)
Article number054112
JournalBiomicrofluidics
Volume13
Issue number5
DOIs
StatePublished - Sep 1 2019

ASJC Scopus subject areas

  • Biomedical Engineering
  • Materials Science(all)
  • Condensed Matter Physics
  • Fluid Flow and Transfer Processes
  • Colloid and Surface Chemistry

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