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Related Experiment Video

Updated: Jun 4, 2026

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity
09:33

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Published on: January 5, 2016

Phosphate ethylation interference assay.

Michael Carey, Stephen T Smale

    CSH Protocols
    |March 2, 2011
    PubMed
    Summary
    This summary is machine-generated.

    The ethylation interference assay identifies DNA-binding protein sites by using ethyl nitrosourea to modify DNA phosphates. This modification, when analyzed with specific protein concentrations, reveals critical DNA-protein interaction sites.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • DNA-protein interactions are fundamental to cellular processes.
    • Identifying specific DNA binding sites for proteins is crucial for understanding gene regulation and function.
    • Existing methods may have limitations in precision or scope.

    Purpose of the Study:

    • To detail the methodology and critical parameters of the ethylation interference assay.
    • To enable precise identification of DNA recognition sites for DNA-binding proteins.
    • To provide a reliable method for mapping protein-DNA interfaces.

    Main Methods:

    • Utilizing ethyl nitrosourea for controlled ethylation of DNA phosphates.
    • Employing (32)P-end-labeled DNA probes containing protein recognition sites.

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  • Incubating modified DNA with specific, barely saturating concentrations of DNA-binding proteins.
  • Analyzing piperidine-induced cleavage products on polyacrylamide/urea gels alongside a sequencing ladder.

    • Main Results:

    • Optimized ethylation yields approximately one ethyl group per DNA molecule, avoiding multiple modifications.
    • Ethylated phosphates interfere with protein binding near the minor groove or phosphate backbone.
    • Differential analysis of cleavage products distinguishes modified DNA with and without bound protein, pinpointing the recognition site.

    Conclusions:

    • The ethylation interference assay is a robust method for mapping DNA-protein binding sites.
    • Precise control over ethylation extent and protein concentration is key to assay success.
    • This technique facilitates detailed characterization of DNA-binding protein interactions.