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.

Figure 1. Comparison of GST-pulldown methodologies: microfuge tube versus filter-plate protocols.

Non-recombinant GST, GST-SMRTτ (amino acids 2077-2471; A, C, E and F), or GST-ACTR (amino acids 621-821; B, D, and F) bound to glutathione agarose were incubated with in vitro translated ³⁵S-methionine radiolabeled TRα1 (A, B and F), RARα (C and D), or FXR (E) protein in either the absence (open bars) or presence (filled bars) of cognate agonist (1 µM T3 for TRα1, 1µM ATRA for RARα or 100 µM CDCA for FXR). Samples were bound, washed, and eluted with free reduced glutathione using the conventional individual tube assay (“Single Tube”), the modified filter microplate assay (“Filter Plate”), or the glutathione coated Reacti-Bind plate assay (“Coated Plate”). Extended wash filter microplate samples (“Extnd. Wash”) were incubated 15 min between washes. Samples were resolved on a SDS-10% PAGE gel prior to fixing, staining and scanning of the dried gel with a Molecular Dynamics Storm 840 Phosphorimager. Quantification of the radiolabeled bands was performed using ImageQuant software version 4.2. Assays were performed in triplicate; error bars indicate standard deviation. (F) Representative Phosphorimager gel images used for analysis in panels A and B.

Figure 2. Titration of the binding of regulators to nuclear receptors by the plate or single tube methods.

GST-ACTR (amino acids 621-821), either 400 ng (triangles) or 200 ng (diamonds), bound to glutathione agarose was incubated with increasing amounts of in vitro translated ³⁵S-methionine radiolabeled TRα1 (A) or RARα (B) in the presence of cognate agonist (1 µM T3 for TRα or 1 µM ATRA for RARα). GST-SMRTτ (amino acids 2077-2471) bound to glutathione agarose was incubated with increasing amounts of in vitro translated ³⁵S-methionine radiolabeled TRα1 (C) in the absence of cognate agonist. Samples were bound, washed, and eluted with free reduced glutathione in either the conventional individual tube assay (squares) or the modified filter microplate assay (triangles) and were analyzed as in Figure 1. Data was fit to a single-site hyperbolic binding curve using GraphPad Prism v. 4.0. (D) The Phosphorimager gel images used for analysis in panel C.

Figure 3. Analysis of the effects of hormone agonists on the receptor/coactivator interaction using the plate method.

GST-ACTR (amino acids 621-821) bound to glutathione agarose was incubated with in vitro translated ³⁵S-methionine radiolabeled TRα1 (A) or RARα (B) protein in presence of increasing amounts of cognate agonist. Samples were analyzed using the filter plate method as in Figure 1. Data was fit to a sigmoidal dose response curve with variable slope using GraphPad Prism v. 4.0.

Figure 4. Comparison of centrifuge washing versus vacuum manifold washing for plate methodology.

(A) GST-SMRTτ (amino acids 2077-2471) bound to glutathione agarose was incubated with in vitro translated ³⁵S-methionine radiolabeled TRα1 protein; empty wells, glutathione agarose alone, or glutathione agarose bound to GST were used as negative controls as indicated. Samples were washed using either a centrifuge (open bars) or vacuum manifold (filled bars), eluted and analyzed as described in Figure 1. (B) GST-ACTR (amino acids 621-821) bound to glutathione agarose was incubated with in vitro translated ³⁵S-methionine radiolabeled TRα1 protein in either the absence or presence of cognate agonist (1 µM T3). Samples were washed using either a centrifuge (open bars) or vacuum manifold (filled bars), eluted, and analyzed using the filter plate method as described in Figure 1. Assays were performed in triplicate; error bars indicate standard deviation.

Figure 5. Comparison of plate assay quantification by SDS-PAGE/phosphorimager analysis versus analysis by liquid scintillation counting.

Non-recombinant GST or GST-ACTR (amino acids 621-821) were bound to glutathione agarose, and were incubated with in vitro translated ³⁵S-methionine radiolabeled TRα1 or RARα protein in presence of cognate agonist (1 µM T3 or 1 µM ATRA). Samples were washed and eluted using the filter plate method as described in Figure 1. After elution, a 20 µl aliquot (out of 70 µl total) of each sample was resolved on a SDS-10% PAGE gel prior to Coomassie staining and visualization of the dried gel with a Molecular Dynamics Storm 840 Phosphorimager. A separate 5 µl aliquot of each sample was subjected to scintillation counting using BioSafe II scintillation cocktail (Research Products International, Mt. Prospect, IL) and a Beckman LS6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Assays were performed in triplicate; error bars indicate standard deviation.