Protein-protein interaction assays: eliminating false positive interactions

Nguyen, Tuan N.; Goodrich, James A.
February 2006
Nature Methods;Feb2006, Vol. 3 Issue 2, p135
Academic Journal
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 interaction. 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 nuclease. Micrococcal nuclease (also known as S7 nuclease) cleaves single- and double-stranded DNA and RNA with no sequence specificity. 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.


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