Anti-Aryl hydrocarbon Receptor antibody [RPT9] - ChIP Grade (ab2769)

Overview

  • Product name
    Anti-Aryl hydrocarbon Receptor antibody [RPT9] - ChIP Grade
    See all Aryl hydrocarbon Receptor primary antibodies
  • Description
    Mouse monoclonal [RPT9] to Aryl hydrocarbon Receptor - ChIP Grade
  • Specificity
    This antibody specifically immunoprecipitates a single ~95 kDa protein representing AHR from Hepa 1 cytosol. Immunohistochemical staining of AHR in rat liver results in strong cytoplasmic and some nuclear staining.
  • Tested applications
    Suitable for: ICC/IF, ELISA, WB, IHC-Fr, ChIP, IHC-P, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Rabbit
  • Immunogen

    Recombinant fragment corresponding to Mouse Aryl hydrocarbon Receptor aa 12-31.
    Sequence:

    R(12)KRRKP(17) V(22)KPIPAEGIK(31)

  • Positive control
    • rat liver tissue sections Hepa 1 cytosolic lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab2769 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
ICC/IF 1/10 - 1/100.
ELISA Use at an assay dependent concentration.
WB 1/500 - 1/2000. PubMed: 21266776
IHC-Fr Use at an assay dependent concentration. PubMed: 23036591
ChIP 1/100.
IHC-P Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
Flow Cyt Use 1-5µg for 106 cells.

Target

  • Function
    Ligand-activated transcriptional activator. Binds to the XRE promoter region of genes it activates. Activates the expression of multiple phase I and II xenobiotic chemical metabolizing enzyme genes (such as the CYP1A1 gene). Mediates biochemical and toxic effects of halogenated aromatic hydrocarbons. Involved in cell-cycle regulation. Likely to play an important role in the development and maturation of many tissues.
  • Tissue specificity
    Expressed in all tissues tested including blood, brain, heart, kidney, liver, lung, pancreas and skeletal muscle.
  • Sequence similarities
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Cellular localization
    Cytoplasm. Nucleus. Initially cytoplasmic; upon binding with ligand and interaction with a HSP90, it translocates to the nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Ah receptor antibody
    • AhR antibody
    • AHR_HUMAN antibody
    • Aromatic hydrocarbon receptor antibody
    • Aryl hydrocarbon receptor antibody
    • Aryl hydrocarbon receptor precursor antibody
    • bHLHe76 antibody
    • Class E basic helix loop helix protein 76 antibody
    • Class E basic helix-loop-helix protein 76 antibody
    • HGNC:348 antibody
    see all

Images

  • Western blot analysis of Aryl Hydrocarbon Receptor was performed by loading 40 ug of HEK293 lysate overexpressing Aryl Hydrocarbon Receptor (right lane) or empty vector control (left lane) and 10ul of a Prestained Protein Ladder onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Aryl Hydrocarbon Receptor monoclonal antibody (ab2769) at a dilution of 1:1000 overnight at 4°C on a rocking platform then washed in TBS-0.1%Tween-20 and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed.

  • Immunocytochemistry/Immunofluorescence analysis of Aryl Hydrocarbon Receptor shows staining in A2058 cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2769 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Aryl Hydrocarbon Receptor shows staining in HeLa cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2769 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Aryl Hydrocarbon Receptor shows staining in U251 cells. Aryl Hydrocarbon Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2769 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • ICC/IF image of ab2769 stained HepG2 cells. The cells were methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2769, in a 1/200 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • This was a trial experiment to evaluate the association of AhR to the ABCG2 promoter using ab2769 at a 1/100 dilution for the IP (ChIP assay). Semiquantitative PCR was performed to evaluate the relative association of AhR with the proximal ABCG2 promoter in a S1 colon cancer cell line without treatment, or treated with depsipeptide (10 ng/mL 24h) or benzo(a)pyrene (10uM 24h). Cross-linking (X-ChIP) - 10 Mins 0 Secs
    Lane 1: DNA ladder (100 bp from promega)
    Inputs (lanes 2-4): S1 no treatment - serial dilution
    Inputs (lanes 5-7): S1 treated with depsipeptide - serial dilution
    Inputs (lanes 8-10): S1 treated with benzo(a)pyrene - serial dilution
    AhR ChIP (lane 11-13): use 1:1 diluted immunoprecipitate for PCR
    Lane 11=S1 no treatment lane 12=S1 depsipeptide 10ng/mL lane 13= S1 benzo(a)pyrene 10uM
    AhR ChIP (lane 14-16): use 1:2 diluted immunoprecipitate for PCR
    same order as lanes 11-13
    Lane 17: H2O control for PCR.
  • ab2769 staining Aryl hydrocarbon Receptor in Mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/750) for 1 hour. Ab98784 (1/500) was used as the secondary antibody. Background staining due to secondary with positive stainind seen in the cytoplasm of the hepatocytes

    See Abreview

  • Flow cytometry analysis of Aryl Hydrocarbon Receptor showing positive staining in the nucleus and cytoplasm of NIH/3T3 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2769 at 1:50 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Aryl Hydrocarbon Receptor showing positive staining in the nucleus and cytoplasm of PC-3 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2769 at 1:50 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Aryl Hydrocarbon Receptor showing positive staining in the nucleus and cytoplasm of Hela cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2769 at 1:50 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Overlay histogram showing HEK293 cells stained with ab2769 (red line). The cells were fixed with 100% 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 (ab2769, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti mouse-DyLight® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Adesso S  et al. Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia. Front Pharmacol 8:370 (2017). IF ; Mouse . Read more (PubMed: 28659803) »
  • Stanford EA  et al. The role of the aryl hydrocarbon receptor in the development of cells with the molecular and functional characteristics of cancer stem-like cells. BMC Biol 14:20 (2016). ChIP . Read more (PubMed: 26984638) »

See all 16 Publications for this product

Customer reviews and Q&As


For ab2769, ChIP was added to the datasheet based on customer feedback received by the originating lab:

1. ChIP assay: The cross-linked, sheared chromatin solution was used for immunoprecipitation with 5 μl of anti-AHR antibody ab2...

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Thank you for providing this information.

I am not aware of these two products (ab2769 and ab2770) being tested against one another in an effort to determine which is better suited for Western Blot. Generally when looking for a product to ...

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Thank you for your reply.
I did send an email to you on Friday afternoon, and I do apologize if you did not receive it. There may have been an issue on our end sending the email. Below is a copy of the email that was sent:
"Based on the informa...

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Thank you for calling Abcam earlier today.

I am sorry about the issues you have been having with ab2769 in western blot. Please let me know if the protocol variations that we talked about do not prove to be successful and I will be happy to re...

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Thank you for contacting Abcam.
Based on the information that you have provided, I do not think that there is any protocol advice that I could provide that would reoslve the issue. Therefore, as the antibody is covered under our Abpromise, I would ...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Liver)
Specification
Liver
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: pH 6.0 Citrate
Permeabilization
No
Username

Abcam user community

Verified customer

Submitted May 24 2012

Thank you for contacting Abcam
The buffer for ab2769 is PBS with 0.05% sodium azide.
Please let me know if there is anything else I can help you with.

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - other (brain capillary membranes)
Loading amount
10 µg
Specification
brain capillary membranes
Treatment
Roche Diagnostics Complete Protease Inhibitor
Gel Running Conditions
Reduced Denaturing (Invitrogen NuPage Bis-Tris 4-12%, 1 mm x 15 wells)
Blocking step
Pierce Protein-free T20 (TBS) Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Mar 02 2012

Successful immunoprecipitations were performed with the antibody using Protein A/G agarose affinity resin and antibody concentrations of 5-10 ug mixed with 400 ug of rat brain lysate. The reference below also details an IP application that was done wit...

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Application
Western blot
Sample
Mouse Cell lysate - whole cell (embryonic stem cell)
Loading amount
200000 cells
Specification
embryonic stem cell
Gel Running Conditions
Reduced Denaturing (10% gel)
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C
Username

Abcam user community

Verified customer

Submitted Dec 17 2010

1-10 of 17 Abreviews or Q&A

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