Journal
/
Biochemistry
/
Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
JoVE Journal
Biochemistry
A subscription to JoVE is required to view this content.  Sign in or start your free trial.
JoVE Journal Biochemistry
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

•

08:27 min

•

January 07, 2019

•

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

Chapters

  • 00:04Title
  • 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

Automatic Translation

Automatic Translation

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

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

check_url/58754?article_type=v

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
check_url/58754?article_type=v

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy
check_url/58754?article_type=v

Single Particle Cryo-Electron Microscopy: From Sample to Structure
check_url/58754?article_type=v

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy
check_url/58754?article_type=v

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows
check_url/58754?article_type=v

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion
check_url/58754?article_type=v

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination
check_url/58754?article_type=v

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon
check_url/58754?article_type=v

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy
check_url/58754?article_type=v

Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes (Video) | JoVE

Read Article