Progesterone Receptor, human membrane suspension

Catalog Number R9907 Storage Temperature –70 °C

Synonym: PGR Procedure Standard Receptor Binding Assay Product Description 1. Prepare Assay Buffer – 25 mM Tris-HCl containing Progesterone regulates reproductive function through 1.5 mM EDTA, 25 mM Na molybdate, and 1 mM two intracellular receptors: an amino-terminal truncated DTT, pH 7.4. form known as progesterone receptor-A (PRA) and 2. Thaw product vial quickly and mix with Assay progesterone receptor-B (PRB). These arise from a Buffer. Resuspend 100 units of receptor in 20 mL of single gene and function as transcriptional regulators of Assay Buffer. Homogenize and store on ice until progesterone responsive genes. hPRB functions as a addition to assay tubes. transcriptional activator, while hPRA is transcriptionally 3. Prepare Radioligand Solution – 6 nM inactive due to its inability to efficiently recruit [3H] Promegestone. coactivators (GRIP1 and SRC-1) upon agonist binding. 4. Prepare Unlabeled Ligand Solution – 10 mM hPRA functions as a strong ligand-dependent Promegestone. transdominant repressor of steroid hormone receptor 5. Prepare the assay volume by combining 200 mL of transcriptional activity. An inhibitory domain (ID) within receptor suspension, 25 mL of Radioligand Solution human PR, which is necessary for transrepression by (0.6 nM final), and 25 mL of Unlabeled Ligand hPRA, has been identified. Progesterone receptors are Solution (1000 nM final). expressed in normal human mammary tissue, 6. Incubate for 18–22 hours at 4 °C. endometrium, hypothalamus, and in cancers of the 1-3 7. Use a dextran coated charcoal capture of the breast and astrocytomas. unbound ligand. 8. Add the assay volume to a 96 well plate. Up to Progesterone Receptor, human is a frozen aliquot of 100 assays may be performed with the solutions membranes from human breast carcinoma cells. Each prepared. vial contains 100 units of receptor membrane suspension at a receptor density of ~290 fmoles/mg Results protein. The membrane suspensions are supplied at Typical results of Standard Receptor Binding Assay. ~11 mg/mL in 50 mM Tris-HCl containing 1.5 mM Results may vary from lot to lot. EDTA, 25 mM Na molybdate, and 1 mM DTT, pH 7.4. Progesterone (h) Saturation Analysis Precautions and Disclaimer 300 This product is for R&D use only, not for drug, Kd= 4.6 nM household, or other uses. Please consult the Material Bmax= 286 fmol/mg protein Safety Data Sheet for information regarding hazards 200 Scatchard and safe handling practices. 40

30 protein Storage/Stability 100 B/F 20 10 Store the product tightly sealed at –70 °C. The receptor 0 0 100 200 remains active for several months when stored at Specific Bound, fmol/mg Spec. Bound 0 –70 °C. 0 5 10 15 20 25 3 Repeated freeze-thaw of this product is not [ H] Promegestone, nM recommended. References 1. Chiang, C-H., et al., Hormonal regulation of 3. Giangrande, P.H., et al., The opposing estrogen receptor alpha and beta gene expression transcriptional activities of the two isoforms of the in human granulosa-luteal cells in vitro. J. Clin. human progesterone receptor are due to differential Endocr. Metab., 85, 3828-3839 (2000). cofactor binding. Mol. Cell. Biol., 20, 3102-3115 2. Boonyaratanakornkit, V., et al., Progesterone (2000). receptor contains a proline-rich motif that directly BKR,MAM 05/10-1 interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol. Cell, 8, 269-280 (2001).

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