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.
  
  
  

Androgen Receptor HCA

Assay principle:

  1. 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
  2. F2H®-BHK cells express components of the protein-protein interaction platform, which recruits GFP-tagged AR-LBD to a specific location in the nucleus
  3. AR-LBD-GFP and AR-NTD-RFP constructs are co-transfected into the F2H®-BHK cells
  4. 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
  5. 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:

AR-LBD:

  • wt
  • W741L
  • F876L
  • T877A
  • F876L-T877A

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).

 

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.

Time frames:
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

Ordering
information

Prices in EUR

ProductCodePriceBuy
F2H®-Kit AR-N/C wt (Androgen Receptor, wt) 
  • F2H®-BHK Cell Line, 5x106 cells
  • Platform-ARLBD(wt)-ARNTD, 100µg
  • Positive Control, 30µg
 
f2hk-ar-wt990 EUR Cart
Platform-ARLBD(wt)-ARNTD, 100µg f2h-ar-wt375 EURCart
Platform-ARLBD(W741L)-ARNTD, 100µgf2h-ar-w741l375 EURCart
Platform-ARLBD(F876L)-ARNTD, 100µgf2h-ar-f876l375 EURCart
Platform-ARLBD(T877A)-ARNTD, 100µgf2h-ar-t877a375 EURCart
Platform-ARLBD(F876L/T877A)-ARNTD, 100µgf2h-ar-f876l/t877a375 EURCart
Positive Control, 30µgf2h-ca85 EUR Cart

Prices in USD

ProductCodePriceBuy
F2H®-Kit AR-N/C wt (Androgen Receptor, wt) 
  • F2H®-BHK Cell Line, 5x106 cells
  • Platform-ARLBD(wt)-ARNTD, 100µg
  • Positive Control, 30µg
 
f2hk-ar-wt1385 USD Cart
Platform-ARLBD(wt)-ARNTD, 100µg f2h-ar-wt525 USDCart
Platform-ARLBD(W741L)-ARNTD, 100µgf2h-ar-w741l525 USDCart
Platform-ARLBD(F876L)-ARNTD, 100µgf2h-ar-f876l525 USDCart
Platform-ARLBD(T877A)-ARNTD, 100µgf2h-ar-t877a525 USDCart
Platform-ARLBD(F876L/T877A)-ARNTD, 100µgf2h-ar-f876l/t877a525 USDCart
Positive Control, 30µgf2h-ca120 USD Cart
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