HTRF Human Total Estrogen Receptor alpha Detection Kit HTRF®

The HTRF Human Estrogen Receptor-alpha assay quantifies the expression level of Estrogen Receptor-alpha in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Estrogen Receptor-alpha 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 Estrogen Receptor-alpha 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 Estrogen Receptor-alpha HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Human Estrogen Receptor-alpha 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 MCF7 or T47D were seeded in a 96-well culture-treated plate under 25,000 cells / well or 50,000 cell / well respectively in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with Tamoxifen (SERM), Fulvestran (SERD) or ARV-471 (PROTAC) compound for 4h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 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 Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

As expected, Fulvestran and ARV-471 induce a dose-dependent decrease of Estrogen receptor alpha expression, while Tamoxifen have no effect.

The human breast cancer cell line MCF7 were seeded in a 96-well culture-treated plate under 25,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated during 4h with ERD-308, a PROTAC compound based on VHL ligand 1 and Raloxifène. An irrelevant PROTAC, dBET6 (targeting BRD4 and Cereblon) was used  to validate specificity. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 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 Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

As expected, only the PROTAC composed of an Estrogen Receptor Alpha ligand and an E3 ligase ligand provoke the protein degradation, not the subunits or an irrelevant PROTAC. GAPDH, a housekeeping protein was used to monitor the cell viability and validate that only the targeted protein was degraded.

The human breast cancer cell line MCF7 were seeded in a 96-well culture-treated plate under 25,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with the ARV-471 or ERD-308 PROTAC compound to a concentration range of 0.02 nM to 100 nM for 4h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 4 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 Estrogen Receptor Alpha detection reagents were added. The HTRF signal was recorded after an overnight of incubation.

A comparison between the HTRF Human Estrogen Receptor Alpha kit and the detection of the protein by western blot was done. The PROTAC compound induce the dose-dependent degradation of Estrogen Receptor Alpha with a IC50 of 1.3 nM for the ARV-471 and  3.7nM for ERD-308. The Estrogen Receptor Alpha degradation obtained with ERD-308 is stronger than with ARV-471 because there is less protein remaining at the maximum concentration of botch compounds.

The human breast cancer cell line MCF7 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 # 4 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 Estrogen Receptor Alpha detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Human Estrogen Receptor Alpha assay, 1,000 cells / well were enough to detect a significant signal, while 8,100 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Human Estrogen Receptor Alpha assay was 8 times more sensitive than the Western Blot technique.

Estrogen Receptor (ER) is a type of nuclear receptor that is activated by estrogen hormones, like Estradiol (E2). The estrogen hormones cross the cell membrane to bind directly onto the Estrogen Receptor, inducing homodimerization of the Estrogen Receptor and permitting nucleus translocation. The Estrogen Receptor dimers then bind to the Estrogen Response Element (ERE) to lead gene expression, resulting in cell division, proliferation, and differentiation.