NDLI: Protein-protein interaction assays: eliminating false positive interactions

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Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)

Protein-protein interaction assays: eliminating false positive interactions

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Protein-protein interaction assays: eliminating false positive interactions

Content Provider	PubMed Central
Author	Nguyen, Tuan N. Goodrich, James A.
Abstract	Many methods commonly used to identify and characterize interactions between two or more proteins are variations of the immobilized protein-protein interaction assay (for example, glutathione S-transferase (GST) pulldown and coimmunoprecipitation). A potential, and often overlooked, problem with these assays is the possibility that an observed interaction is mediated not by direct contact between proteins, but instead by nucleic acid contaminating the protein preparations. As a negatively charged polymer, nucleic acid (often cellular RNA) can adhere to basic surfaces on proteins, and thereby mediate interactions between an immobilized bait protein and a target protein. The contaminating nucleic acid may cause a false positive result in protein-protein interaction assays or may contribute to general background. Alternatively, in relatively rare cases, the presence of nonspecific nucleic acid can inhibit protein-protein interaction1. In general, contaminating nucleic acid can be especially problematic with proteins under study that naturally bind RNA or DNA (for example, transcription factors), although nucleic acid can mediate apparent protein-protein interactions in other systems as well. A simple and convenient method for decreasing false positives and background owing to contaminating nucleic acid is to treat the protein preparations with micrococcal nuclease1–3. Micrococcal nuclease (also known as S7 nuclease) cleaves single- and double-stranded DNA and RNA with no sequence specificity4. This protocol describes a GST pulldown assay that incorporates micrococcal nuclease treatment of both the immobilized GST-bait protein and the target protein preparations. The strategy can be adapted easily to proteins with other tags and immobilized using other methods. The target protein can be derived from many different sources—for example, a cell extract containing a native protein of interest; a recombinant protein expressed in Escherichia coli, insect cells or mammalian cells; a protein produced in an in vitro translation system; or a highly purified protein. The steps required to treat protein preparations with micrococcal nuclease are straightforward and can be incorporated into any immobilized protein-protein interaction assay.
Related Links	http://dx.doi.org/10.1038/nmeth0206-135
Starting Page	135
File Format	PDF
ISSN	15487105
e-ISSN	15487105
Journal	Nature methods
Issue Number	2
Volume Number	3
Language	English
Publisher Date	2006-02-01
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology Biotechnology

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