The Progesterone Receptor (PR) is a nuclear hormone receptor activated by the steroid hormone progesterone. PR regulates gene expression involved in reproductive tissue function, cellular differentiation, and proliferation. It exists in two isoforms, PR-A and PR-B, each with distinct and sometimes opposing roles in gene regulation. PR is a critical player in breast cancer development and is a target for endocrine therapies.
The AlphaLISA SureFire Ultra Human Phospho-Progesterone Receptor (Ser190) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-Progesterone Receptor (Ser190) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Increase of Progesterone receptor in 17-ß estradiol treated cells
MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 0.78 µM of 17-ß Estradiol for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Progesterone Receptor Phospho (Ser190) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, treatment with 17-ß Estradiol caused an increase in both Phospho and Total Progesterone Receptor levels.
Increase of Progesterone receptor in Promegestone treated cells
MCF7 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO2. The cells were treated with 1 µM of Promegestone for 2 hours. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Progesterone Receptor Phospho (Ser190) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays.
For the detection step, 10 µL of cell lysate (approximately 2,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, treatment with Promegestone, a well published progesterone receptor agonist, caused an increase in both Phospho (Ser190) and Total Progesterone Receptor levels.
Decrease of Progesterone receptor in fulvestrant treated cells
MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 10 µg/mL of Fulvestrant for 18 hours. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Progesterone Receptor Phospho (Ser190) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays.
For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Fulvestrant triggered a decrease of both Phospho (Ser190) and Total Progesterone Receptor while Total Cofilin levels remained unchanged (data not shown).
