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Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
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JoVE Journal Biyokimya
Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

DOI:
10.3791/59038-v

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10:50 min

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March 14, 2019

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, ,
1Department of Biochemistry,Purdue Üniversitesi, 2Department of Botany and Plant Pathology,Purdue Üniversitesi, 3Center for Plant Biology,Purdue Üniversitesi

Bölümler

  • 00:04Başlık
  • 00:33Designing the FRET Assay
  • 01:12Preparation of Cul1AMC•Rbx1, the FRET Donor Protein
  • 04:42Preparation of FlAsHCand1, the FRET Acceptor Protein
  • 05:18Testing and Confirmation of the FRET Assay
  • 06:08Measuring the Association Rate Constant
  • 07:37Measuring the Dissociation Rate Constant of Cul1•Cand1 in the Presence of Skp•F-box Protein
  • 08:33Results: Dynamics Of Protein Complexes
  • 10:30Conclusion

Özet

Otomatik Çeviri

Otomatik Çeviri

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique.  

Etiketler

FRETProtein ComplexKineticsAssociationDissociationCOL1CAND17-amino-4-methylcoumarinFlASHCell LysisSonicationCentrifugationGlutathione BeadsElutionThrombinBuffer A

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