An improved high throughput protein-protein interaction assay for nuclear hormone receptors.
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.
Goodson
Michael L
ML
Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California, USA.
Farboud
Behnom
B
Privalsky
Martin L
ML
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
D018160:Receptors, Cytoplasmic and Nuclear; D005982:Glutathione Transferase