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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
JoVE Journal
Bioquímica
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JoVE Journal Bioquímica
Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

DOI:
10.3791/61401-v

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08:33 min

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March 11, 2021

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, ,
1Department of Physiology, Anatomy and Genetics,University of Oxford, 2Department of Chemistry and Neuroscience Program,Trinity College

Capítulos

  • 00:04Introduction
  • 00:36Unroofed Membrane Experiments
  • 04:36Patch-Clamp Fluorometry Experiment
  • 05:56Results: Representative Measurement of Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
  • 07:55Conclusion

Summary

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This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as Förster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Tags

Nucleotide BindingMembrane ProteinsFRETATPPotassium ChannelDiabetesUnroofed MembraneSonicationMicroscopyANAPFluorescence Imaging

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