Journal
/
Biochemistry
/
Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization
JoVE Journal
Biochemistry
This content is Free Access.
JoVE Journal Biochemistry
Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

DOI:
10.3791/60747-v

•

09:19 min

•

March 16, 2020

•

, , , , , ,
1Laboratory of Biomolecular Research,Paul Scherrer Institute, 2Department of Biology,ETH Zürich, 3Center for Radiopharmaceutical Sciences,Paul Scherrer Institute, 4Laboratory of Molecular Biology,Medical Research Council

Chapters

  • 00:04Introduction
  • 00:45Cell Membrane Solubilization and Protein Extraction
  • 03:53UV-VIS Spectroscopy
  • 04:33Automated Size-Exclusion Chromatography of Rhodopsin and Rhodopsin Mini-Go Complex
  • 06:04Deglycosylation
  • 06:44Results: UV-VIS Spectroscopy, SEC, and SDS-PAGE
  • 08:34Conclusion

Summary

Automatic Translation

Automatic Translation

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

Tags

Keywords: GPCRMini-G ProteinSignal TransductionMembrane Protein ComplexDetergent ScreeningCrystallizationHEK293 CellsAffinity Purification9-cis RetinalBuffer Exchange

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

check_url/60747?article_type=v

Protein Complex Affinity Capture from Cryomilled Mammalian Cells
check_url/60747?article_type=v

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
check_url/60747?article_type=v

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
check_url/60747?article_type=v

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering
check_url/60747?article_type=v

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization
check_url/60747?article_type=v

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening
check_url/60747?article_type=v

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
check_url/60747?article_type=v

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex
check_url/60747?article_type=v

High-Throughput Protein Crystallization via Microdialysis
check_url/60747?article_type=v

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography (Video) | JoVE

Read Article