Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-IRF3 antibody [EPR2418Y] - BSA and Azide free (ab201809)

Overview

  • Product name

    Anti-IRF3 antibody [EPR2418Y] - BSA and Azide free
    See all IRF3 primary antibodies
  • Description

    Rabbit monoclonal [EPR2418Y] to IRF3 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

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

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human IRF3 aa 50-150. The exact sequence is proprietary.
    Database link: Q14653

  • Positive control

    • WB: HeLa, Jurkat, THP-1, Daudi, HepG2 whole cell lysates. Human fetal heart and kidney lysates. Mouse heart and spleen lysates, NIH/3T3 whole cell lysates. IHC-P: Human tonsil, Human squamous cell carcinoma of cervix, Mouse spleen. ICC/IF: HeLa cells Flow: U937 cells
  • General notes

    ab201809 is the carrier-free version of ab68481 This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Ab201809 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab201809 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-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB Use at an assay dependent concentration. Detects a band of approximately 51 kDa (predicted molecular weight: 47 kDa).
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

  • Application notes
    Is unsuitable for IP.
  • 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

    • Overlay histogram showing HAP1 wildtype (green line) and HAP1-IRF3 knockout cells (red line) stained with ab68481. The cells were fixed with 80% methanol (5 min) (left pannel) or 4% formaldehyde (10 min) (right pannel), and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab68481, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

      A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-IRF3  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

      Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

      Note: We recommend fixing cells using MeOH instead of PFA to get optimal results.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    • Flow cytometry analysis of 2% paraformaldehyde fixed U937 (Human histiocytic lymphoma cells) cells labeling IRF3 with ab68481 at 1/160 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling IRF3 with ab68481 at 1/100 dilution. Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/400 dilution was used as the secondary antibody (green). The confocal image shows cytoplasmic on HeLa cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
      The negative controls are as follows;
      1. ab68481 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
      2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

       

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    • Immunohistochemical analysis of paraffin-embedded Mouse spleen labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    • Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    • Immunohistochemical analysis of paraffin-embedded Human tonsil labeling IRF3 with ab68481at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68481).

    References

    This product has been referenced in:

    • Meng X  et al. C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1. J Virol 86:4538-47 (2012). WB . Read more (PubMed: 22345458) »
    • Liu F  et al. Klotho suppresses RIG-I-mediated senescence-associated inflammation. Nat Cell Biol 13:254-62 (2011). Read more (PubMed: 21336305) »
    See all 2 Publications for this product

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