HTRF Human Androgen Receptor Detection Kit HTRF®
The Human Androgen Receptor kit is designed to monitor the expression level of cellular Androgen Receptor.
The HTRF Human Androgen Receptor detection assay monitors Wild-Type Androgen Receptor (AR-WT) and the Androgen Receptor splice Variant 7 (AR-V7), and is used to detect the expression of endogenous or overexpressed Androgen Receptors in various cells.
Androgen Receptor is a type of nuclear receptor that is activated by androgen hormones, like testosterone (T) and Dihydrotestosterone (DHT).
Androgens bind to the ligand binding domain of the Androgen Receptor, resulting in receptor homodimerization and translocation to the nucleus. This activates downstream gene expression and activation of signaling pathways, like the PI3K/AKT pathway. The gene expression induced by Androgen Receptor results in cell division, proliferation, differentiation, apoptosis, and angiogenesis. A dysregulation of the Androgen Receptor pathway could lead to oncogenic phenotypes, and Androgen Receptor activation or surexpression has been widely linked to prostate cancer as well as other types of cancer, like breast cancer and glioblastoma. Moreover, Androgen Receptor variants are expressed in various human cancer cell lines. The Androgen Receptor Variant 7 (AR-V7), a splice variant of the androgen receptor mRNA resulting in the truncation of the ligand-binding domain, is constitutively active and widely expressed in human cancer cell lines. Androgen Receptor has therefore become an important drug target for the pharmaceutical industry. The development of PROTAC compounds to target Androgen Receptor degradation is a part of the therapeutic strategy to target Androgen Receptor positive cancers.
The HTRF Human Androgen Receptor assay quantifies the expression level of Androgen Receptor in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Androgen Receptor assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Androgen Receptor in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Human Androgen Receptor HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Human Androgen Receptor with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
The human breast cancer cell line MDA-MB453 was cultured in a T175 flask in complete culture medium for 48h at 37 ° C, 5% CO2. The cells were lyzed with 3 mL of supplemented lysis buffer # 3 for 30 minutes at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Human Androgen Receptor detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.
Using the HTRF Human Androgen Receptor assay, 1,500 cells / well were enough to detect a significant signal, while 6,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Human Androgen Receptor assay was 4.5 times more sensitive than the Western Blot technique.
The human breast cancer cell line MDA-MB453 or the human prostate cancer cell line LNCaP were seeded in a 96-well culture-treated plate under 75,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with the ARCC-4 PROTAC compound to a concentration range of 0.1 nM to 10 000 nM for 24h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 3 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Human Androgen Receptor detection reagents were added. The HTRF signal was recorded after 2h of incubation.
A comparison between the HTRF Human Androgen Receptor kit and the detection of AR-WT and AR-V7 by western blot was done. The ARCC-4 PROTAC compound induce the dose-dependent degradation of Androgen Receptor with a IC50 of 56 nM for the MDA-MB453 cell line and 911 nM for the LNCaP cell line. The Androgen Receptor degradation obtained with the HTRF Human Androgen Receptor kit highligted the degradation of the WT form of AR (110 KDa). Indeed the two cell lines illustrated expressed the WT and V7 forms of AR. Considering the missing ligand binding domain of the V7 form of AR (80 kDa), the ARCC-4 PROTAC compound can not bind to the AR-V7 and thus do not induce the AR-V7 degradation.
Androgen Receptor (AR) is a type of nuclear receptor that is activated by androgen hormones, like testosterone (T) and dihydrotestosterone (DHT). The androgen hormones cross the cell membrane to bind directly onto the Androgen Receptor, inducing homodimerization of the Androgen Receptor and permiting nucleus translocation. The Androgen Receptor dimers then bind to the Androgen Response Element (ARE) to lead gene expression, resulting in cell division, proliferation, differentiation, apoptosis, and angiogenesis.
physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Notes techniques
HTRF and WB compatible guidelines - Notes techniques
Protocol for tumor xenograft analysis with HTRF - Notes techniques
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Notes d'application
Tumor xenograft analysis: HTRF versus Western blot - Notes d'application
Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application
Increased flexibility of phospho-assays - Notes d'application
Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application
In collaboration with Bayer - Présentations scientifiaues
A fun video introducing you to phosphorylation assays with HTRF - Vidéos
Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques
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