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Chromotek/F2H®-Kit Androgen Receptor Analyze modulators of the human Androgen Receptor
Detect early steps of Androgen Receptor activation
Detect early steps of Androgen Receptor activation
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Chromotek/F2H®-Kit Androgen Receptor
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Chromotek/F2H®-Kit Androgen Receptor
Analyze modulators of the human Androgen Receptor
Detect early steps of Androgen Receptor activation
Visualized in live mammalian cells
Real-time agonists and antagonists screening
Convenient readout using an epi-fluorescence microscope
The human Androgen receptor (AR) is an important target for developing drug therapies combating prostate cancer. The very early event in AR activation, namely the intramolecular interaction between the N- and C-terminus of AR, can now be analyzed with the ChromoTek Fluorescent-Two Hybrid (F2H®) AR: wild-type (wt), as well as several mutant AR forms can be investigated.
Chromotek/F2H®-Kit Androgen Receptor
Assay principle:
AR is expressed as two separate domains: AR-LBD (ligand binding domain), comprising the AR’s C-terminus, either wt or mut, fused to GFP: AR-LBD-GFP
AR-NTD (N-terminal domain), comprising the AR’s N-terminus, fused to RFP: AR-NTD-RFP
F2H®-BHK cells express components of the protein-protein interaction platform, which recruits GFP-tagged AR-LBD to a specific location in the nucleus
AR-LBD-GFP and AR-NTD-RFP constructs are co-transfected into the F2H®-BHK cells
AR-LBD-GFP forms a bright green spot at the specific location in the nuclei whereas AR-NTD-RFP is diffusely distributed in the nucleus
Upon stimulation by an agonist (e.g. DHT), immobilized AR-LBD-GFP undergoes a conformation change and recruits AR-NTD-RFP: AR-NTD-RFP forms a bright red spot superimposed with the green spot.
Results:
Green spot is present, red spot is absent => assay works: no interaction
Green spot is present, red spot is present at the same location => assay works: interaction
Figure: AR-LBD-GFP (top left) forms bright green spots at the genetic Platform in nuclei of F2H-BHK cells. Before addition of DHT, AR-NTD-RFP (bottom left) is dispersed in the nuclei. Upon 20 min incubation with 10nM DHT, the red spots appear (bottom right, arrows) at the location of the green spots, indicating induction of the interaction between AR-LBD and AR-NTD.
Reversible Screening:
The F2H AR assay is fully reversible: the AR wt interaction can be induced by an agonist (e.g. 0.25nM DHT) and then subsequently disrupted by addition of a potent antagonist (e.g. 10µM bicalutamide).