Journal
/
Biochimie
/
Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
Journal JoVE
Biochimie
Un abonnement à JoVE est nécessaire pour voir ce contenu.  Connectez-vous ou commencez votre essai gratuit.
Journal JoVE Biochimie
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

•

10:49 min

•

September 20, 2016

•

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

Chapitres

  • 00:05Titre
  • 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

Traduction automatique

Traduction automatique

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

Article

Embed

AJOUTER À LA PLAYLIST

Usage Statistics

Vidéos Connexes

check_url/fr/54529?article_type=v

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
check_url/fr/54529?article_type=v

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
check_url/fr/54529?article_type=v

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis
check_url/fr/54529?article_type=v

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins
check_url/fr/54529?article_type=v

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy
check_url/fr/54529?article_type=v

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase
check_url/fr/54529?article_type=v

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes
check_url/fr/54529?article_type=v

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
check_url/fr/54529?article_type=v

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence
check_url/fr/54529?article_type=v

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Read Article