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Rensing av Native Komplekser for Structural Study Ved hjelp av en Tandem Affinity Tag Method
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Journal JoVE Biologie
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Rensing av Native Komplekser for Structural Study Ved hjelp av en Tandem Affinity Tag Method

DOI:
10.3791/54389-v

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10:36 min

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July 27, 2016

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1Department of Biochemistry,Brandeis University, 2Winship Cancer Institute,Emory University School of Medicine

Chapitres

  • 00:05Titre
  • 00:38Harvesting S. cerevisiae Cells
  • 01:44Lyse Cells and Lysate Clarification
  • 04:43Resin Equilibration and Binding and On Column TEV Cleavage
  • 06:47Packing and Eluting from Calmodulin Resin
  • 08:20Results: TAP Purification of U1 snRNP Complex in S. cerevisiae
  • 09:55Conclusion

Summary

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Traduction automatique

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Tags

Native Macromolecular ComplexPurificationTandem Affinity TagStructural StudyS. CerevisiaeCell LysisLiquid NitrogenCoffee GrinderCentrifugationBuffer ConditionsProtein Complex

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