Western blot analysis of extracts from T-47D cells grown for 48 hr in phenol red-free medium supplemented with 5% charcoal-stripped FBS, untreated (-) or promegestone (R5020)-treated (100 nM, 1 hr; +), using Phospho-Progesterone Receptor (Ser294) Antibody (upper) and Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb #8757 (lower).
|MW (kDa)||90 (PR-A), 118 (PR-B)|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Phospho-Progesterone Receptor (Ser294) Antibody recognizes endogenous levels of progesterone receptor B (PR-B) and progesterone receptor A (PR-A) proteins only when phosphorylated at Ser294 and Ser130, respectively. This antibody does not cross-react with other progesterone receptor family members.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Pig, Horse
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser294 of human progesterone receptor B (PR-B) protein. Antibodies are purified by protein A and peptide affinity chromatography.
Human progesterone receptor (PR) is expressed as two forms: the full length PR-B and the short form PR-A. PR-A lacks the first 164 amino acid residues of PR-B (1,2). Both PR-A and PR-B are ligand activated, but differ in their relative ability to activate target gene transcription (3,4). The activity of PR is regulated by phosphorylation; at least seven serine residues are phosphorylated in its amino-terminal domain. Three sites (Ser81, Ser102, and Ser162) are unique to full length PR-B, while other sites (Ser190, Ser294, Ser345, and Ser400) are shared by both isoforms (5). Phosphorylation of PR-B at Ser190 (equivalent to Ser26 of PR-A) is catalyzed by CDK2 (6). Mutation of Ser190 results in decreased activity of PR (7), suggesting that the phosphorylation at Ser190 may be critical to its biological function.
Progesterone receptor Ser294 is as an important hormone-dependent phospho-acceptor site that serves as an extracellular signaling “sensor." Research studies indicate that p44/p42 MAP kinases are responsible for phosphorylation of progesterone receptor at Ser294 following hormone binding. This phosphorylation event promotes nuclear retention of the receptor, enhanced transcriptional activity, and ubiquitin-dependent proteasomal degradation (6,8,9).
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