Anti-Progesterone Receptor antibody [PR-AT 4.14] - ChIP Grade (ab2764)

Overview

  • Product nameAnti-Progesterone Receptor antibody [PR-AT 4.14] - ChIP GradeSee all Progesterone Receptor primary antibodies ...
  • Description
    Mouse monoclonal [PR-AT 4.14] to Progesterone Receptor - ChIP Grade
  • SpecificityDetects progesterone receptor. This antibody does not cross-react with androgen receptor, estrogen receptor or glucocorticoid receptor.
  • Tested applicationsIHC-Fr, IP, WB, IHC-P, Gel supershift assays, ChIP, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Guinea pig, Cow, Dog, Human, Marsupials, Rhesus monkey
  • Immunogen

    Synthetic peptide corresponding to Human Progesterone Receptor aa 533-547.
    Sequence:

    GLPQVYPPYLNLRP


    (Peptide available as ab4907)

  • Positive control
    • Rat uterus can be used for IHC.

Properties

Applications

Our Abpromise guarantee covers the use of ab2764 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr Use a concentration of 5 µg/ml. Immunohistochemical staining of PR in rat uterus with this antibody yields intense nuclear staining.
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 99 kDa.Can be blocked with Progesterone Receptor peptide (ab4907). By Western blot, this antibody detects a 94 kDa and a 120 kDa protein representing PRA and PRB, respectively from calf uterine homogenate.
IHC-P Use a concentration of 5 µg/ml.
Gel supershift assays Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration. PubMed: 19553525
ICC/IF 1/500. Using T47D cells.
Flow Cyt Use 0.1-1µg for 106 cells.

Target

  • FunctionThe steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Progesterone receptor isoform B (PRB) is involved activation of c-SRC/MAPK signaling on hormone stimulation.
    Isoform A: inactive in stimulating c-Src/MAPK signaling on hormone stimulation.
    Isoform 4: Increases mitochondrial membrane potential and cellular respiration upon stimulation by progesterone.
  • Sequence similaritiesBelongs to the nuclear hormone receptor family. NR3 subfamily.
    Contains 1 nuclear receptor DNA-binding domain.
  • DomainComposed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.
  • Post-translational
    modifications
    Phosphorylated on multiple serine sites. Several of these sites are hormone-dependent. Phosphorylation on Ser-294 occurs preferentially on isoform B, is highly hormone-dependent and modulates ubiquitination and sumoylation on Lys-388. Phosphorylation on Ser-102 and Ser-345 also requires induction by hormone. Basal phosphorylation on Ser-81, Ser-162, Ser-190 and Ser-400 is increased in response to progesterone and can be phosphorylated in vitro by the CDK2-A1 complex. Increased levels of phosphorylation on Ser-400 also in the presence of EGF, heregulin, IGF, PMA and FBS. Phosphorylation at this site by CDK2 is ligand-independent, and increases nuclear translocation and transcriptional activity. Phosphorylation at Ser-162 and Ser-294, but not at Ser-190, is impaired during the G(2)/M phase of the cell cycle. Phosphorylation on Ser-345 by ERK1/2 MAPK is required for interaction with SP1.
    Sumoylation is hormone-dependent and represses transcriptional activity. Sumoylation on all three sites is enhanced by PIAS3. Desumoylated by SENP1. Sumoylation on Lys-388, the main site of sumoylation, is repressed by ubiquitination on the same site, and modulated by phosphorylation at Ser-294.
    Ubiquitination is hormone-dependent and represses sumoylation on the same site. Promoted by MAPK-mediated phosphorylation on Ser-294.
    Palmitoylated by ZDHHC7 and ZDHHC21. Palmitoylation is required for plasma membrane targeting and for rapid intracellular signaling via ERK and AKT kinases and cAMP generation.
  • Cellular localizationNucleus. Cytoplasm. Nucleoplasmic shuttling is both homone- and cell cycle-dependent. On hormone stimulation, retained in the cytoplasm in the G(1) and G(2)/M phases; Mitochondrion outer membrane and Nucleus. Cytoplasm. Mainly nuclear.
  • Information by UniProt
  • Database links
  • Alternative names
    • NR3C3 antibody
    • NR3C3 antibody
    • Nuclear receptor subfamily 3 group C member 3 antibody
    • PGR antibody
    • PGR antibody
    • PR antibody
    • PR antibody
    • PRA antibody
    • PRB antibody
    • PRGR_HUMAN antibody
    • Progesterone receptor antibody
    • Progestin receptor form A antibody
    • Progestin receptor form B antibody
    see all

Anti-Progesterone Receptor antibody [PR-AT 4.14] - ChIP Grade images

  • Overlay histogram showing T47D cells stained with ab2764 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2764, 1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
  • T47D cells were treated with progesterone (10X-8M) for 2 hours, and then fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/1000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-mouse Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 timT47D cells were treated with progesterone (10X-8M) for 2 hours, and then fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/1000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-mouse Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab2764, blue staining by DAPI.
  • ab2764 at a 1/400 dilution staining Progesterone Receptor in rat uterus tissue by Immunohistochemistry.
    Tissue was fixed in formaldehyde and permeabilized using 0.3% Triton-X. 10% serum was used in the blocking step. The secondary used was a Goat anti-mouse conjugated to biotin, 1/500 dilution.

    This antibody worked well in rat sections. However, in mouse sections, due to very poor homology between the immunogen used and the mouse nPR receptor, the antibody did not work despite taking steps to prevent mouse-on-mouse staining issues. So in short, this antibody is good for rat sections, where immunogen homology is better, but be careful about using it in mouse sections.
    ab2764 at a 1/400 dilution staining Progesterone Receptor in rat uterus tissue by Immunohistochemistry.
    Tissue was fixed in formaldehyde and permeabilized using 0.3% Triton-X. 10% serum was used in the blocking step. The secondary used was a Goat anti-mouse conjugated to biotin, 1/500 dilution.

    This antibody worked well in rat sections. However, in mouse sections, due to very poor homology between the immunogen used and the mouse nPR receptor, the antibody did not work despite taking steps to prevent mouse-on-mouse staining issues. So in short, this antibody is good for rat sections, where immunogen homology is better, but be careful about using it in mouse sections.
  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized Human uterus tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Progesterone Receptor ab2764 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

References for Anti-Progesterone Receptor antibody [PR-AT 4.14] - ChIP Grade (ab2764)

This product has been referenced in:
  • Bondi CD  et al. The effect of estradiol, progesterone, and melatonin on estrous cycling and ovarian aromatase expression in intact female mice. Eur J Obstet Gynecol Reprod Biol 174:80-5 (2014). WB ; Mouse . Read more (PubMed: 24373455) »
  • Yu Y  et al. Prostate stromal cells express the progesterone receptor to control cancer cell mobility. PLoS One 9:e92714 (2014). IHC ; Human . Read more (PubMed: 24664419) »

See all 16 Publications for this product

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Thank you for your reply.

I can confirm the following concentrations:

ab2764 Progesterone Receptor antibody [PR-AT 4.14] is sold at 1 mg/ml
ab9269 Estrogen Receptor alpha antibody [6F11] is sold at 75 ug/ml

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I can confirm that ab2764 antibody is sold as ascites. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet. Antib...

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The antibody has been tested with cow samples so it is fully guarnateed. Please find attahecd the supporting image.

I hope this information is helpful to you. Please do not hesitate to contact us if you ...

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I can confirm that we recommend not to dilute antibodies before storage unless is states to do so on the individual datasheet. For all antibodies, we suggest to always follow instructions provided on the individua...

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Unseres Wissens haben wir noch keine Abbildung von ab2764 in WB mit Ratten Proben. Falls Sie uns eine Abbildung Ihrer Ergebnisse mit ab2764 zukommen lassen, ka...

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Products;

PAX6; http://www.abcam.com/PAX6-antibody-Stem-Cell-Marker-ab5790.html

NeuN; http://www.abcam.com/FOX3NeuN-antibody-ab104225.html

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http://www.abcam.com/index.htm...

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Thank you for your reply and for pointing that out. I have updated the note on our website to reflect the correct species.

I have been in touch with the lab and they did not have an image of rat lysate in WB using this antibody. They were onl...

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By Western blot, this antibody detects a 94 kDa and a 120 kDa protein representing PRA and PRB, respectively from rat uterine homogenate. (PubMed: 16912069). They refer to our internal catalogue # MA1-410, but th...

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I am sorry this product did not perform as stated on the datasheet and for the inconvenience this...

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