Anti-IRF3 antibody [EP2419Y] (ab76409)

Knockout Tested Rabbit monoclonal IRF3 antibody [EP2419Y]. Validated in WB, IP, IHC, Flow Cyt and tested in Human. Cited in 9 publication(s).

Overview

  • Product name

    Anti-IRF3 antibody [EP2419Y]
    See all IRF3 primary antibodies
  • Description

    Rabbit monoclonal [EP2419Y] to IRF3
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IP, IHC-P, Flow Cytmore details
    Unsuitable for: ICC
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human IRF3 (C terminal). The exact sequence is proprietary.

  • Positive control

    • U937, HeLa, MCF7 and Jurkat cell lysates; human tonsil tissue.
  • General notes

     

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information. 

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76409 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
WB 1/1000 - 1/2000. Predicted molecular weight: 47 kDa.
IP 1/20.
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
Flow Cyt 1/10.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

  • Application notes
    Is unsuitable for ICC.
  • Target

    • Function

      Mediates interferon-stimulated response element (ISRE) promoter activation. Functions as a molecular switch for antiviral activity. DsRNA generated during the course of an viral infection leads to IRF3 phosphorylation on the C-terminal serine/threonine cluster. This induces a conformational change, leading to its dimerization, nuclear localization and association with CREB binding protein (CREBBP) to form dsRNA-activated factor 1 (DRAF1), a complex which activates the transcription of genes under the control of ISRE. The complex binds to the IE and PRDIII regions on the IFN-alpha and IFN-beta promoters respectively. IRF-3 does not have any transcription activation domains.
    • Tissue specificity

      Expressed constitutively in a variety of tissues.
    • Sequence similarities

      Belongs to the IRF family.
      Contains 1 IRF tryptophan pentad repeat DNA-binding domain.
    • Post-translational
      modifications

      Constitutively phosphorylated on many serines residues. C-terminal serine/threonine cluster is phosphorylated in response of induction by IKBKE and TBK1. Ser-385 and Ser-386 may be specifically phosphorylated in response to induction. An alternate model propose that the five serine/threonine residues between 396 and 405 are phosphorylated in response to a viral infection. Phosphorylation, and subsequent activation of IRF3 is inhibited by vaccinia virus protein E3.
      Ubiquitinated; ubiquitination involves RBCK1 leading to proteasomal degradation. Polyubiquitinated; ubiquitination involves TRIM21 leading to proteasomal degradation.
      ISGylated by HERC5 resulting in sustained IRF3 activation and in the inhibition of IRF3 ubiquitination by disrupting PIN1 binding. The phosphorylation state of IRF3 does not alter ISGylation.
    • Cellular localization

      Cytoplasm. Nucleus. Shuttles between cytoplasmic and nuclear compartments, with export being the prevailing effect. When activated, IRF3 interaction with CREBBP prevents its export to the cytoplasm.
    • Information by UniProt
    • Database links

    • Alternative names

      • IIAE7 antibody
      • Interferon regulatory factor 3 antibody
      • IRF 3 antibody
      • IRF-3 antibody
      • IRF3 antibody
      • IRF3_HUMAN antibody
      • MGC94729 antibody
      see all

    Images

    • Lane 1: Jurkat cell lysate (20 µg)

      Lane 2: MCF7 cell lysate (20 µg)

      Lane 3: HeLa wildtype cell lysate (20 µg)

      Lane 4: IRF3 HeLa knockout cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab76409 observed at 47 kDa. Red - loading control, ab8245 observed at 37 kDa.

      ab76409 was shown to react with IRF3 in HeLa wildtype. Loss of signal was observed when knockout sample ab263784 was used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab76409 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
    • Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: IRF3 knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: Jurkat cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab76409 observed at 50 kDa. Red - loading control, ab8245, observed at 37kDa.


      ab76409 was shown to react with IRF3 in wild-type HAP1 cells alond with additional cross-reactive bands. No band was observed when IRF3 knockout samples were used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab76409 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

    • All lanes : Anti-IRF3 antibody [EP2419Y] (ab76409) at 1/2000 dilution

      Lane 1 : U937 cell lysate
      Lane 2 : Hela cell lysate
      Lane 3 : MCF7 cell lysate
      Lane 4 : Jurkat cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 47 kDa
      Observed band size: 47 kDa

    • Immunohistochemical analysis of IRF3 in paraffin embedded human tonsils using ab76409 at a 1/100 dilution.

      Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

    • Flow cytometric analysis of permeabilized U937 cells using anti- IRF-3 RabMAb (red) (ab76409) or a rabbit IgG (negative) (green)

    References

    This product has been referenced in:

    • Tajpara P  et al. A Preclinical Model for Studying Herpes Simplex Virus Infection. J Invest Dermatol 139:673-682 (2019). Read more (PubMed: 30414908) »
    • Reisländer T  et al. BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors. Nat Commun 10:3143 (2019). Read more (PubMed: 31316060) »
    See all 12 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Answer

    Thank you for contacting us.

    We have published safety datasheets for these products on the website. These will be available soon. Please download these from our website.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Answer

    Thank you once again for your recent telephone enquiry. I am pleased to forward the testing flow cytometry protocol for ab76409 IRF3 antibody [EP2419Y] from the originator. Please note, this is a general testing protocol and may require some optimization in your own laboratories. Solutions and reagents 1. 1X PBS buffer 2. Blocking buffer: 0.5% BSA in 1X PBS 3. 2% paraformaldehyde (1% solution – optional for storing samples) 4. 1X FACS permeabilizing solution 5. Fluorescently conjugated secondary antibody Protocol 1. Collect 1 X 106 cells/sample 2. Wash cells once with blocking buffer 3. Fix cells with 2% paraformaldehyde and incubate at room temperature for 10 min 4. Wash cells once with blocking buffer 5. Add 0.5ml 1X FACS permeabilizing solution and incubate at room temperature for 10 min 6. Wash cells once with blocking buffer 7. Incubate cells in blocking buffer for 30 min at room temperature 8. Add primary antibody at the appropriate dilution and incubate for 30 min at room temperature 9. Wash twice with blocking buffer and incubate with fluorescently conjugated secondary antibody for 20 min at room temperature 10. Wash cells twice with blocking buffer 11. Re-suspend cells in 1X PBS and analyze on flow cytometer. Samples can be kept in 1% paraformaldehyde at 4oC overnight I hope this will be helpful. Should you have any further questions, please do not hesitate to contact us.

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

    Sign up