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Biochemistry

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

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

Article DOI: 10.3791/61401-v • 08:33 min • March 11th, 2021
March 11th, 2021 •
1, 1, 1,2
1Department of Physiology, Anatomy and Genetics, University of Oxford, 2Department of Chemistry and Neuroscience Program, Trinity College

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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.

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Nucleotide Binding Intact Membrane Proteins Real-time Measurement FRET Fluorescently Tagged Proteins Spatial Resolution Cellular Metabolism ATP Sensitive Potassium Channel Diabetes Unroofed Membrane Experiment Cover Slip Transfected Cells PBS Polylysine Coating Probe Sonicator Amplitude Unroofing Cells Bath Solution Inverted Microscope
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