Validated using a knockout cell line

Anti-IRF5 antibody [10T1] (ab33478)

Reviews (3)Q&A (2)References (14)

Overview

  • Product name

    Anti-IRF5 antibody [10T1]
    See all IRF5 primary antibodies

  • Description

    Mouse monoclonal [10T1] to IRF5

  • Host species

    Mouse

  • Specificity

    ab33478 recognises IRF-5.

  • Tested applications

    Suitable for: ICC/IF, WB, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant human IRF-5 (176-240 aa, isoform D) purified from E. coli. NCBI Accession No.: NP_001092099 (formally NP_002191).

  • Positive control

    • WB: Extracts of Ramos, THP-1 and A20 cells. ICC/IF: THP-1, Raw264.7 and HEK293. FC: THP-1 cells.

  • General notes

    This product was changed from ascites to tissue culture supernatant on 24/01/2019. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab33478 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 Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Involvement in disease

    Genetic variations in IRF5 are associated with susceptibility to inflammatory bowel disease type 14 (IBD14) [MIM:612245]. IBD14 is a chronic, relapsing inflammation of the gastrointestinal tract with a complex etiology. It is subdivided into Crohn disease and ulcerative colitis phenotypes. Crohn disease may affect any part of the gastrointestinal tract from the mouth to the anus, but most frequently it involves the terminal ileum and colon. Bowel inflammation is transmural and discontinuous; it may contain granulomas or be associated with intestinal or perianal fistulas. In contrast, in ulcerative colitis, the inflammation is continuous and limited to rectal and colonic mucosal layers; fistulas and granulomas are not observed. Both diseases include extraintestinal inflammation of the skin, eyes, or joints.
    Genetic variations in IRF5 are associated with susceptibility to systemic lupus erythematosus type 10 (SLEB10) [MIM:612251]. Systemic lupus erythematosus (SLE) is a chronic, inflammatory and often febrile multisystemic disorder of connective tissue. It affects principally the skin, joints, kidneys and serosal membranes. It is thought to represent a failure of the regulatory mechanisms of the autoimmune system.
    Genetic variations in IRF5 are a cause of susceptibility to rheumatoid arthritis (RA) [MIM:180300]. It is a systemic inflammatory disease with autoimmune features and a complex genetic component. It primarily affects the joints and is characterized by inflammatory changes in the synovial membranes and articular structures, widespread fibrinoid degeneration of the collagen fibers in mesenchymal tissues, and by atrophy and rarefaction of bony structures.

  • Sequence similarities

    Belongs to the IRF family.
    Contains 1 IRF tryptophan pentad repeat DNA-binding domain.

  • Cellular localization

    Nucleus.
  • Information by UniProt

  • Database links

    • Alternative names

      • Interferon regulatory factor 5 antibody
      • Interferon regulatory factor 5 bone marrow variant antibody
      • IRF 5 antibody
      • IRF-5 antibody
      • Irf5 antibody
      • IRF5_HUMAN antibody
      • SLEB10 antibody
      see all

    Images

    • Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/500 dilution

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : IRF5 knockout HAP1 whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 58 kDa



      Lanes 1 - 2: Merged signal (red and green). Green - ab33478 observed at 56 kDa. Red - loading control, ab181602, observed at 37 kDa.

      ab33478 was shown to specifically react with IRF5 in wild-type HAP1 cells as signal was lost in IRF5 knockout cells. Wild-type and IRF5 knockout samples were subjected to SDS-PAGE. Ab33478 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

      This image was produced using the ascites version of this antibody. 

    • Flow Cytometry - Anti-IRF5 antibody [10T1] (ab33478)
      Flow Cytometry - Anti-IRF5 antibody [10T1] (ab33478)

      Flow cytometry analysis of IRF5 in THP-1 cell line, staining at 2-5µg for 1x106 cells (red line). The secondary antibody used goat anti-mouse IgG Alexa fluor 488 conjugate. Isotype control antibody was mouse IgG (black line).

    • Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)
      Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)
      Immunocytochemistry/ Immunofluorescence analysis of IRF5 in Raw264.7 cells. The cell was stained with ab33478 (1:100). The secondary antibody (green) was used Alexa Fluor 488. DAPI was stained the cell nucleus (blue).
    • Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)
      Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)
      Immunocytochemistry/ Immunofluorescence analysis of IRF5 in THP-1 cells. The cell was stained with ab33478 (1:100). The secondary antibody (green) was used Alexa Fluor 488. DAPI was stained the cell nucleus (blue).
    • Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      Anti-IRF5 antibody [10T1] (ab33478) at 1/1000 dilution + THP-1 cell lysate

      Secondary
      Goat anti-mouse secondary antibody conjugated to HRP

      Predicted band size: 58 kDa

    • Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)
      Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [10T1] (ab33478)

      Immunofluorescence/Immunocytochemistry analysis of HEK293 cells (105-106) stained with Mouse monoclonal [10T1] to IRF5 (ab33478 1/100-1/200).

      This image was produced using the ascites version of this antibody. 

    • Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      Western blot - Anti-IRF5 antibody [10T1] (ab33478)
      All lanes : Anti-IRF5 antibody [10T1] (ab33478) at 1/1000 dilution

      Lane 1 : Ramos cell lysate
      Lane 2 : THP-1 cell lysate
      Lane 3 : A20 cell lysate
      Lane 4 : NIH 3T3 cell lysate

      Secondary
      All lanes : goat anti-mouse secondary antibody conjugated to HRP

      Developed using the ECL technique.

      Predicted band size: 58 kDa
      Observed band size: 58 kDa
      Additional bands at: 26 kDa, 80 kDa. We are unsure as to the identity of these extra bands.



      100-200µg of whole cell lysates were used for western blotting.

      This image was produced using the ascites version of this antibody. 

    • Flow Cytometry - Anti-IRF5 antibody [10T1] (ab33478)
      Flow Cytometry - Anti-IRF5 antibody [10T1] (ab33478)

      Overlay histogram showing THP1 cells stained with ab33478 (red line). The cells were fixed with 4% paraformaldehyde (10 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. The cells were then incubated with the antibody (ab33478, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 THP1 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

      This image was produced using the ascites version of this antibody. 

    References

    This product has been referenced in:

    • Moen SH  et al. Human Toll-like Receptor 8 (TLR8) Is an Important Sensor of Pyogenic Bacteria, and Is Attenuated by Cell Surface TLR Signaling. Front Immunol 10:1209 (2019). Read more (PubMed: 31214180) »
    • Xiao Y  et al. An oligodeoxynucleotide with AAAG repeats significantly attenuates burn-induced systemic inflammatory responses via inhibiting interferon regulatory factor 5 pathway. Mol Med 23:N/A (2017). Read more (PubMed: 28620671) »
    See all 14 Publications for this product

    Publishing research using ab33478? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-5 of 5 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-IRF5 antibody [10T1]

     Excellent
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Macrophages)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Ventana Ultra CC1
    Permeabilization
    No
    Specification
    Macrophages
    Blocking step
    OptiView H2O2 as blocking agent for 8 minute(s) · Concentration: 0.04% · Temperature: 36°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 25 2016

    Western blot abreview for Anti-IRF5 antibody [10T1]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    30 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Human Cell lysate - whole cell (macrophage)
    Specification
    macrophage
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 12 2014

    Question

    Phone call reporting problems in using anti-IRF5 antibody [10T1] (ab33478) in western blotting.

    Read More
    Answer

    Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

    As we discussed on the phone, I have attachedour questionnaire so that we can gather further information regarding the samples tested and the protocol used.This information will also allow us to investigate this case internally and initiate additional testing where necessary.

    As discussed over the phone, I have arranged for a replacement vial of ab33478 to be sent out to you. This should reach you tomorrow or Friday. The order number is xxxxxx (Purchase order number FOCR xxxxx). If you do not find that your results improve with this new lot please do let me know.

    I look forward to receiving your reply.

    Read More

    Immunohistochemistry (Frozen sections) abreview for Anti-IRF5 antibody [10T1]

     Average
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain)
    Specification
    Brain
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - heat, Triton X-100
    Blocking step
    Sniper (BioCare) as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 25°C
    Read More

    Ms. Colleen Karlo

    Verified customer

    Submitted Oct 04 2011

    Question

    I have a few questions about your anti-IRF5 antibody, cat.#:ab33478. Q1: what is the suggested concentration for detection of IRF5 in human dendritic cells? Q2: In the web site, the band shown in lane 2 is the endogenous IRF5 or inducible IRF5 in THP-1 cells? If it is inducible, what is the inducer and the concentration? Q3: Is ab6728 is the cat.# for HRP antibody used to show the IRF5 band? Q4: Dose milk or BSA used in blocking buffer make any differences in showing IFR5 expression? I would greatly appreciate your promptly response.

    Read More
    Answer

    Thank you for your interest in our product, anti-IRF5 antibody(ab33478). I now have some more details which should answer your questions. 1. The anti-IRF5 Antibody (ab33478) was used at 1:1,000 dilution, and 30~50mg protein of cell lysates was used for detection in western blotting. 2. Bands shown in the data sheet are endogenous IRF5, they are not inducible protein. 3. A commercialised secondary antibody, goat anti-mouse Ig-HRP conjugated was used. This was not ab6728. 4. You can get same results for milk or BSA as blocking solution, So you may use anything. I hope that theses answers are helpful for you and please contact me again if you have any other questions. With thanks.

    Read More

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