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Protein Complex Affinity Capture from Cryomilled Mammalian Cells
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

DOI:
10.3791/54518-v

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10:37 min

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December 09, 2016

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, ,
1Laboratory of Cellular and Structural Biology,The Rockefeller University, 2Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology,New York University School of Medicine

Chapters

  • 00:05Title
  • 00:30Cell Harvesting and Freezing
  • 03:09Cryogenic Disruption of Frozen Cells
  • 04:50Affinity Capture of Protein Complexes from Cell Extracts
  • 06:02Affinity Capture
  • 07:41Results: Comparing Some Different Aspects of Affinity Capture Optimization
  • 09:38Conclusion

Summary

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Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Tags

Keywords: Protein ComplexAffinity CaptureCryomilledMammalian CellsCell BiologyMolecular BiologyProtein InteractionsPBSCell PelletLiquid NitrogenCell Lysis

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