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Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO
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Biochemistry
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JoVE Journal Biochemistry
Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

DOI:
10.3791/60339-v

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13:02 min

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December 28, 2019

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, , ,
1Department of Chemistry,Dartmouth College

Chapters

  • 00:04Title
  • 01:12Large Scale Expression of Histidine-6 (His6)-Tagged Nuclear Factor-kappa-B Essential Modulator Crystallography Construct (NEMO-EEAA)
  • 02:33His6-Tagged NEMO-EEAA Purification
  • 04:42His6 Tag Cleavage and Purification
  • 07:09Sparse Matrix Screening
  • 08:23Seed Stock Generation
  • 09:00Crystal Looping
  • 09:43X-Ray Data Processing and Structure Solution
  • 10:38Results: Representative Production, Crystallization, and Structure Determination of the IKK-Binding Domain of NEMO
  • 12:28Conclusion

Summary

Automatic Translation

Automatic Translation

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

Tags

NEMOIKK-binding DomainProtein ProductionCrystallizationStructure DeterminationCoiled-coil AdaptersProtein RefoldingProtein ConcentrationAggregationSolubilityFrench PressCell LysisUreaProtein Purification

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