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Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry
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Biochimica
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JoVE Journal Biochimica
Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

DOI:
10.3791/62107-v

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07:27 min

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April 09, 2021

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, ,
1Department of Biology,University of Waterloo

Capitoli

  • 00:04Introduction
  • 00:44Cell Culture
  • 01:25Preparation and Saponification of Fatty Acid Analogs
  • 03:35Click Reaction on Cell Lysates
  • 04:12Click Reaction on Immunoprecipitated Proteins
  • 05:13Results: Detection of Fatty Acylated Proteins Using Click Chemistry
  • 06:57Conclusion

Summary

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The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

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Keywords: Alkynyl Fatty Acid AnalogsClick ChemistryFatty Acylated ProteinsFatty Acid LabelingMetabolic LabelingHEK-293 T-cellsBSA-bound Fatty AcidsSaponificationPotassium HydroxideFatty Acid-free BSA

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