Progesterone Receptor, phosphorylated, Human (NR3C3, PGR) BioAssay(TM) ELISA Kit

The progesterone Receptor (PR; NR3C3) is a member of the steroid receptor superfamily which binds progesterone. The progesterone receptor is expressed in uterus, ovary, vagina, fallopian tubes and breast. Progesterone receptors play a central role in diverse reproductive events associated with the establishment and maintenance of pregnancy, alveolar development in the breast, and sexual behavior. Two isoforms of the progesterone receptor have been identified, progesterone receptor-A (PR-A) and progesterone receptor-B (PR-B), 94 and 114kD in size, respectively. The 'B' progesterone receptor (PR-B), or long form, is 933 amino acids (aa) in length. Progesterone receptor-A (PR-A) utilizes a different start site that shortens the N-terminus by 164 amino acids. The N-terminus in both progesterone receptor isoforms is rich in serine that is phosphorylated in response to hormone binding. In the absence of progesterone, few receptor molecules are phosphorylated at Serine 190 (S190). Progesterone increases this number two-fold, providing evidence for hormone stimulation. It has been shown that the two progesterone receptor isoforms can form homodimers as well as heterodimers.\n\nSample Type:\nWhole cells\n\nIntended Use:\nThis Cell-Based ELISA BioAssay(TM) contains the components required to run an ELISA using fluorogenic substrates to measure Progesterone Receptor (PR) phosphorylated at S294 in whole cells. This simple and efficient assay eliminates the need to prepare cell lysates and can be used to investigate signaling pathways and the effects of inhibitors on cells.\n\nSpecificity:\nThis antibody specifically recognizes human Progesterone Receptor isoform A and B, and cross-reacts with rat Progesterone Receptor.\n\nTest Principle:\nCells are grown in 96-well plates and stimulated with ligands. Following stimulation, cells are fixed and permeabilized in the wells. The target protein phosphorylation is measured using a double immunoenzymatic labeling procedure. The cells are simultaneously incubated with two primary antibodies: a phospho-specific antibody and a normalization antibody that recognizes GAPDH, a house keeping protein. The primary antibodies are derived from different species. Two secondary antibodies recognizing the different species are labeled with either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for either HRP or AP are used for detection. The fluorescence of the phosphorylated protein is normalized to that of GAPDH in each well for the correction of well-to-well variations. This two-wavelength assay results in precise analysis of protein phosphorylation with good reproducibility.\n\nKit Components:\nPhospho-PR (S294) Antibody, 1x vial \nTotal GAPDH (Ms) Antibody, 1x vial\nHRP-conjugated anti-rabbit IgG, 1x110ul\nAP-conjugated anti-mouse IgG, 1x110ul\nSubstrate F1 Concentrate, 1x50ul\nF1 Diluent, 1x10ml\nSubstrate F2, 1x10ml\nBlocking Buffer, 1x35ml\nWash Buffer (5X), 1x60ml\nMicroplate, 1x96wells\nPlate Sealers, 4x adhesive strips\n\nStorage and Stability:\nStore powder at 4 degrees C liquid at -20 degrees C. Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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