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.
- 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.
Scheme: AR-LBD-GFP and AR-NTD-RFP fusion proteins interact at the genomic Platform in F2H-BHK cells upon stimulation with DHT
Available AR variants:
- 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.
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).
Movie: Live-cell F2H imaging of reversible interactions between AR-LBD-GFP (green) and AR-NTD-RFP (red) in F2H-BHK cells upon 0.25nM DHT and 10µM bicalutamide treatments. The presence of green spots indicate that AR-LBD-GFP has been anchored to the platform.
Before DHT addition – no red spots – no interaction
1 h after DHT addition – red spots appear – interaction between AR-LBD and AR-NTD is induced
2 h after DHT addition, bicatlutamide is just added – red spots present – interactions persist
4 h after DHT addition, 2 h after bicalutamide addition – red spots disappear – interaction is disrupted
Prices in EUR
|F2H®-Kit AR-N/C wt (Androgen Receptor, wt) ||f2hk-ar-wt||990 EUR||Cart|
|Platform-ARLBD(wt)-ARNTD, 100µg||f2h-ar-wt||375 EUR||Cart|
|Platform-ARLBD(W741L)-ARNTD, 100µg||f2h-ar-w741l||375 EUR||Cart|
|Platform-ARLBD(F876L)-ARNTD, 100µg||f2h-ar-f876l||375 EUR||Cart|
|Platform-ARLBD(T877A)-ARNTD, 100µg||f2h-ar-t877a||375 EUR||Cart|
|Platform-ARLBD(F876L/T877A)-ARNTD, 100µg||f2h-ar-f876l/t877a||375 EUR||Cart|
|Positive Control, 30µg||f2h-ca||85 EUR||Cart|
Prices in USD
|F2H®-Kit AR-N/C wt (Androgen Receptor, wt) ||f2hk-ar-wt||1385 USD||Cart|
|Platform-ARLBD(wt)-ARNTD, 100µg||f2h-ar-wt||525 USD||Cart|
|Platform-ARLBD(W741L)-ARNTD, 100µg||f2h-ar-w741l||525 USD||Cart|
|Platform-ARLBD(F876L)-ARNTD, 100µg||f2h-ar-f876l||525 USD||Cart|
|Platform-ARLBD(T877A)-ARNTD, 100µg||f2h-ar-t877a||525 USD||Cart|
|Platform-ARLBD(F876L/T877A)-ARNTD, 100µg||f2h-ar-f876l/t877a||525 USD||Cart|
|Positive Control, 30µg||f2h-ca||120 USD||Cart|