HTRF Human Total Progesterone Detection Kit, 500 Assay Points

Human Total Progesterone Receptor Detection Kit assay principle

The HTRF Human Progesterone Receptor assay quantifies the expression level of Progesterone Receptor in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Progesterone 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 the presence of Progesterone 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.

Human Total Progesterone Receptor Detection Kit two -plate assay protocol

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 Progesterone Receptor HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Human Total Progesterone Receptor Detection Kit one-plate assay protocol

Detection of Human Progesterone 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.

Estrogen targeted compound effect on Estrogen and Progesterone Receptor Expression

The human breast cancer cell line MCF7 was used for this illustration. Cells were seeded in a 96-well culture-treated plate under 50,000 cells/well in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with Tamoxifen (SERM), Fulvestran (SERD), or ERD-308 (PROTAC) compounds for 4h or 16h. After treatment, the cells were lysed with 50 µL of supplemented lysis buffer 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 or HTRF Human Progesterone Receptor detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, Tamoxifen did not have major effect on Estrogen Receptor, but induced a decrease in the expression of Progestrone Receptor. Fulvestran and ERD-308 induced a decrease in Estrogen Receptor alpha and Progesterone Receptor expression. The effects of the compounds were more rapid in Estrogen Receptor compared to their effect on Progesterone Receptor, which is coherent with the literature.

Estrogen Receptor compound treatment in MCF7 cells

Cells from the human breast cancer cell line MCF7 was seeded in a 96-well culture-treated plate under 50,000 cell/well in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with Estradiol (agonist), Tamoxifen (SERM), Fulvestrant (SERD), or ERD-308 (PROTAC) compounds for 16 hours. After treatment, the cells were lysed with 50 µL of supplemented lysis buffer 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 Progesterone Receptor detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, Fulvestrant, ERD308 and Tamoxifen induce a dose-dependent decrease of Progesterone Receptor expression, while Estradiol had no effect.

Progesterone Receptor Signaling Pathway

Progesterone Receptor (PR) is a nuclear receptor that is activated by progesterone. The hormone crosses the cell membrane and binds to the receptor. After binding, PR homodimerizes, enabling translocation to the nucleus. Coupled with a co-activator, the receptor dimer will be recruited to the Progesterone Response Element (PRE) and induce gene expression leading to proliferation / apoptosis balance.

PR expression is modulated by Estrogen Receptor. Therefore, PR is a biomarker commonly used as an indicator of Estrogen Receptor-α function. The MAPK kinase pathway has also been described as having an effect on PR activity through phosphorylation.