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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
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Biochimie
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Journal JoVE Biochimie
Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

DOI:
10.3791/58754-v

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08:27 min

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January 07, 2019

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, , , , ,
1Van Andel Institute, 2Vollum Institute, 3Janelia Research Campus

Chapitres

  • 00:04Titre
  • 00:34Infection of HEK293 Mammalian Cells with P2 Baculovirus for Large-scale Protein Expression
  • 02:42Purification of hTRPC3 Protein from the Frozen Cell Pellet
  • 05:26Results: Structural Determination of hTRPC3
  • 07:36Conclusion

Summary

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Traduction automatique

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Tags

Keywords: TRPC3Ion ChannelProtein ExpressionProtein PurificationSingle-particle Cryo-electron MicroscopyHEK293 CellsVirus InfectionSodium ButyrateDetergent SolubilizationSize-exclusion ChromatographyUltracentrifugation

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