Stoichiometry

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Avtar Singh - One of the best experts on this subject based on the ideXlab platform.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    Scientific Reports, 2020
    Co-Authors: Alexander Song, Avtar Singh, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit Stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    bioRxiv, 2020
    Co-Authors: Avtar Singh, Alexander Song, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex Stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

Warren R. Zipfel - One of the best experts on this subject based on the ideXlab platform.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    Scientific Reports, 2020
    Co-Authors: Alexander Song, Avtar Singh, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit Stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    bioRxiv, 2020
    Co-Authors: Avtar Singh, Alexander Song, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex Stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

Alexander L Van Slyke - One of the best experts on this subject based on the ideXlab platform.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    Scientific Reports, 2020
    Co-Authors: Alexander Song, Avtar Singh, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit Stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    bioRxiv, 2020
    Co-Authors: Avtar Singh, Alexander Song, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex Stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

Maria Sirenko - One of the best experts on this subject based on the ideXlab platform.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    Scientific Reports, 2020
    Co-Authors: Alexander Song, Avtar Singh, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit Stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    bioRxiv, 2020
    Co-Authors: Avtar Singh, Alexander Song, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex Stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

Alexander Song - One of the best experts on this subject based on the ideXlab platform.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    Scientific Reports, 2020
    Co-Authors: Alexander Song, Avtar Singh, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit Stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.

  • stoichiometric analysis of protein complexes by cell fusion and single molecule imaging
    bioRxiv, 2020
    Co-Authors: Avtar Singh, Alexander Song, Maria Sirenko, Paul J. Kammermeier, Alexander L Van Slyke, Warren R. Zipfel
    Abstract:

    The composition, Stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit Stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex Stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex Stoichiometry.