title: An improved high throughput protein-protein interaction assay for nuclear hormone receptors.
abstract: The Glutathione-S-Transferase (GST) "pulldown" assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.
authors.affiliation: Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Goodson
authors.forename: Behnom
authors.initials: B
authors.lastname: Farboud
authors.forename: Martin L
authors.initials: ML
authors.lastname: Privalsky
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D005982:Glutathione Transferase