关键词: ChromoTek—单克隆抗体、重组单域抗体、荧光抗体、GFP或RFP连接蛋白
SERVICE PHONE
4006-400-850
产品中心
PRODUCT CENTER
SERVICE PHONE
4006-400-850

咨询热线

4006-400-850
地址:中国·苏州·生物纳米园
电话:4006-400-850
邮箱:info@moreybio.com

F2H® Assay

当前位置:主页 > 产品中心 > F2H® Assay >

F2H®-Kit Androgen Receptor

发布时间:2017/05/16 点击量:

  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.

  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).

  

 

  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

 

上一篇:F2H®-Kits p53-Mdm2/4

下一篇:没有了

返回