Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA)

K. Roder, M. Schweizer

    Research output: Contribution to journalArticle

    Abstract

    The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition. We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp I (stimulatory protein I), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of glycine concentration on Sp I binding using recombinant glutathione S-transferase-Sp I fusion protein expressed in Escherichia coli.

    Original languageEnglish
    Pages (from-to)209-214
    Number of pages6
    JournalBiotechnology and Applied Biochemistry
    Volume33
    Issue number3
    DOIs
    Publication statusPublished - 2001

    Fingerprint

    Electrophoretic mobility
    Assays
    Buffers
    Gels
    DNA
    Chemical analysis
    Sp Transcription Factors
    Proteins
    Ionic strength
    Glutathione Transferase
    Oligonucleotides
    Glycine
    Escherichia coli
    Nucleic Acids
    Carrier Proteins
    Fusion reactions
    Binding Sites
    RNA
    Temperature

    Keywords

    • Gel retardation
    • GST
    • NF-Y
    • SpI
    • USF

    Cite this

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