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Oligopeptide Competition Assay for Phosphorylation Site Determination
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Biochemistry
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JoVE Journal Biochemistry
Oligopeptide Competition Assay for Phosphorylation Site Determination

Oligopeptide Competition Assay for Phosphorylation Site Determination

DOI:
10.3791/55708-v

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09:16 min

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May 18, 2017

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, , , ,
1College of Pharmacy and Research Institute of Pharmaceutical Sciences,Seoul National University, 2College of Pharmacy and Institute of Pharmaceutical Science and Technology,Hanyang University

Chapters

  • 00:05Title
  • 01:05Site-directed Mutagenesis of pGEX-Nrf2
  • 03:45In Vitro Competitive 5′ Adenosine Monophosphate-activated Protein Kinase (AMPK) Assay
  • 06:13In Vitro AMPK Activity Assay with a Site-specific Mutant
  • 07:39Results: In Vitro AMPK Assays with Competitive Oligopeptides or a Site-specific Mutant Protein
  • 08:31Conclusion

Summary

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Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Tags

Oligopeptide Competition AssayPhosphorylation Site DeterminationCell SignalingPost-translational ModificationsSite-directed MutagenesisPCRDpnI DigestionBacterial TransformationDNA SequencingProtein Expression And Purification

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