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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
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Biochemie
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JoVE Journal Biochemie
Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

DOI:
10.3791/54529-v

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10:49 min

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September 20, 2016

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,
1Department of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, 2Department of Oncology,Johns Hopkins University School of Medicine

Kapitel

  • 00:05Titel
  • 00:43Preparation of Cultured Cells
  • 01:23Treatment of Cells and Photocrosslinking
  • 04:34Attachment of Fluorescent Tag by Click Chemistry for Visualization of Labeled Proteins
  • 05:48Attachment of Biotin Tag by Click Chemistry for Affinity Purification of Labeled Proteins
  • 08:54Results: Live-cell Photoaffinity Labeling Identifies a Major Itraconazole-binding Protein
  • 10:14Conclusion

Summary

Automatische Übersetzung

Automatische Übersetzung

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Tags

Keywords: Small MoleculeBinding ProteinsPhotoaffinity LabelingLive-cellMolecular MechanismDrug TargetProtein IdentificationNative Cellular Environment

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