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High-Throughput Protein Analysis Using Negative Stain Electron Microscopy and 2D Classification

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

High-throughput protein expression and purification allows for fast triaging of several constructs based on expression levels, protein integrity, and solubility. While this technology has been successfully adopted to prioritize constructs for structural biology, it could not inform on important biochemical properties such as domain architecture, homogeneity, and flexibility. Negative staining electron microscopy can be used to quickly evaluate these properties and, if coupled to single particle analysis, can inform on the architecture and conformational state of nearly any protein sample. Here we describe a protocol for negative stain sample preparation, imaging, and two-dimensional (2D) data analysis applicable to a variety of protein complexes. We discuss in more detail a specific application of this technology to large molecule studies to determine the binding sites of individual antibodies on target antigens.

Key words

  • Protein expression
  • Transmission electron microscopy
  • Negative stain EM
  • Single particle analysis
  • 2D classification
  • Epitope mapping

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Correspondence to Christopher P. Arthur or Claudio Ciferri .

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Arthur, C.P., Ciferri, C. (2019). High-Throughput Protein Analysis Using Negative Stain Electron Microscopy and 2D Classification. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_22

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_22

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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