Abstract
In-cell NMR enables structural insights at atomic resolution of proteins in their natural environment. To date, very few methods have been developed to study proteins by in-cell NMR in mammalian systems. Here we describe a detailed protocol to conduct in-cell NMR on the intrinsically disordered protein of alpha-Synuclein (αSyn) in mammalian cells. This chapter includes a simplified expression and purification protocol of recombinant αSyn and its delivery into mammalian cells. The chapter also describes how to assess the cell leakage that might occur to the cells, the setup of the instrument, and how to perform basic analyses with the obtained NMR data.
Keywords:
Electroporation; IDP; Leakage; Nuclear magnetic resonance; α-synuclein.
MeSH terms
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Animals
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Electrophoresis, Polyacrylamide Gel / methods
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Electroporation
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Escherichia coli
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HEK293 Cells
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Humans
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Intrinsically Disordered Proteins / chemistry*
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Isotope Labeling / methods
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Mammals
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Nitrogen Isotopes
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Nuclear Magnetic Resonance, Biomolecular / instrumentation
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Nuclear Magnetic Resonance, Biomolecular / methods*
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Recombinant Proteins / analysis
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Recombinant Proteins / isolation & purification
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Rosaniline Dyes
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Single-Cell Analysis / methods*
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Staining and Labeling / methods
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alpha-Synuclein / analysis
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alpha-Synuclein / chemistry
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alpha-Synuclein / isolation & purification
Substances
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Intrinsically Disordered Proteins
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Nitrogen Isotopes
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Nitrogen-15
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Recombinant Proteins
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Rosaniline Dyes
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alpha-Synuclein
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coomassie Brilliant Blue