使用敲除细胞株进行验证RabMAb

Anti-IRF3抗体[EPR2418Y] (ab68481)

概述

  • 产品名称Anti-IRF3抗体[EPR2418Y]
    参阅全部 IRF3 一抗
  • 描述
    兔单克隆抗体[EPR2418Y] to IRF3
  • 经测试应用适用于: ICC/IF, WB, IHC-P, Flow Cytmore details
    不适用于: IP
  • 种属反应性
    与反应: Mouse, Human
  • 免疫原

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human IRF3 aa 50-150.
    Database link: Q14653

  • 阳性对照
    • 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
  • 常规说明

    This product is a recombinant rabbit monoclonal antibody.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

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

     

    Alternative versions available:

    Anti-IRF3 antibody (Alexa Fluor® 488) [EPR2418Y] (ab204647)

    Anti-IRF3 antibody (Phycoerythrin) [EPR2418Y] (ab209919)

性能

应用

Our Abpromise guarantee covers the use of ab68481 in the following tested applications.

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

应用 Ab评论 说明
ICC/IF 1/100.
WB 1/1000. Detects a band of approximately 51 kDa (predicted molecular weight: 47 kDa).
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Flow Cyt 1/160. ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
  • 应用说明Is unsuitable for IP.
  • 靶标

    • 功能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.
    • 组织特异性Expressed constitutively in a variety of tissues.
    • 序列相似性Belongs to the IRF family.
      Contains 1 IRF tryptophan pentad repeat DNA-binding domain.
    • 翻译后修饰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.
    • 细胞定位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
    • 数据库链接
    • 别名
      • Interferon regulatory factor 3 antibody
      • IRF 3 antibody
      • IRF-3 antibody
      • IRF3 antibody
      • IRF3_HUMAN antibody
      • MGC94729 antibody
      see all

    Anti-IRF3 antibody [EPR2418Y] 图像



    • Predicted band size : 47 kDa

      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 - ab68481 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab68481was shown to specifically react with IRF3 when IRF3 knockout samples were used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and ab8245 (loading control to GAPDH) were both diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    • 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.

       

    • All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

      Lane 1 : Human fetal heart lysate
      Lane 2 : Human fetal kidney lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size : 47 kDa

      Blocking and Dilution buffer: 5% NFDM/TBST

    • All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/10000 dilution

      Lane 1 : THP-1 (Human monocytic leukemia cells) whole cell lysates
      Lane 2 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysates
      Lane 3 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysates

      Lysates/proteins at 10 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

      Predicted band size : 47 kDa
      Observed band size : 51 kDa (why is the actual band size different from the predicted?)

      Blocking and Dilution buffer: 5% NFDM/TBST

    • All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/10000 dilution

      Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
      Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

      Lysates/proteins at 20 µg per lane.

      Secondary
      Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

      Predicted band size : 47 kDa
      Observed band size : 51 kDa (why is the actual band size different from the predicted?)

      Blocking and Dilution buffer: 5% NFDM/TBST

    • All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

      Lane 1 : Mouse heart lysate
      Lane 2 : Mouse spleen lysate
      Lane 3 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

      Lysates/proteins at 10 µg per lane.

      Secondary
      Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

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

      Blocking and Dilution buffer: 5% NFDM/TBST

       

      The slightly smaller molecular mass observed in mouse than in human is supported by literature.

    • 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).

    • 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.

    • 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.

    • 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.

    Anti-IRF3 antibody [EPR2418Y] (ab68481)参考文献

    This product has been referenced in:
    • Wheeler LA  et al. TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice. Cell Rep 15:1715-27 (2016). Read more (PubMed: 27184854) »
    • 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) »

    See all 3 Publications for this product

    Product Wall

    ab68481 has been tested in human hela cells and MCF7 lysates and can be used as positive controls for your experiment to assess the efficacy of the antibody. Abcam provides a whole range of protocols to test both antibodies.

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Flow Cytometry
    Sample Human Cell (primary human macrophages and CD4+ T cells)
    Permeabilization Yes - BD Cytofix/cytoperm kit
    Gating Strategy all live cells
    Specification primary human macrophages and CD4+ T cells
    Preparation Cell harvesting/tissue preparation method: Monocyte-derived macrophages were prepared from human PBMCs CD4+ T cells were separated from human PBMCs
    Sample buffer: 0.5%BSA/PBS
    Fixation Paraformaldehyde
    Username

    Radiana Trifonova

    Verified customer

    提交于 May 27 2016

    ab25950 and ab68481, has been tested in mouse whole cell lysates however,we have only tested the antibody in a reduced, denatured Western blot.
    We are not sure whether it will bind to a dimer in a native Western blot. It is possible, but it's also...

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"