Recombinant
RabMAb

Recombinant Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)

Reviews (1)Q&A (5)References (67)

Overview

  • Product name

    Anti-IRF3 (phospho S386) antibody [EPR2346]
    See all IRF3 primary antibodies

  • Description

    Rabbit monoclonal [EPR2346] to IRF3 (phospho S386)

  • Host species

    Rabbit

  • Specificity

    IRF3 (phospho S386) antibody (ab76493) detects IRF3 when phosphorylated on Serine 386.

  • Tested applications

    Suitable for: WB, Dot blotmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide corresponding to Human IRF3 (phospho S386).
    Database link: Q14653

  • Positive control

    • WB: MCF7 cells treated with Calyculin A.

  • General notes

    A trial size is available to purchase for this antibody.

    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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76493 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 - 1/10000. Predicted molecular weight: 47 kDa.
Dot blot 1/1000.

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

    • Western blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)
      Western blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)
      All lanes : Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493) at 0.2 µg/ml (purified)

      Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
      Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) treated with calyculin A for 45 minutes whole cell lysates
      Lane 3 : MCF7 (Human breast adenocarcinoma epithelial cell) treated with calyculin A for 45 minutes whole cell lysates. Then the membrane was incubated with phosphatase

      Lysates/proteins at 15 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 47 kDa



      Blocking and diluting buffer: 5% NFDM/TBST

      We are unsure to define the extra band at 37KD.

    • Western blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)
      Western blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)
      All lanes : Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493) at 1/20000 dilution (Unpurified)

      Lane 1 : MCF7 cell lysate - treated with Calyculin A
      Lane 2 : MCF7 cell lysate – untreated

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

      Predicted band size: 47 kDa



      Blocking buffer - 5% NFDM/TBST

      Diluting buffer - 1% BSA

    • Dot Blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)
      Dot Blot - Anti-IRF3 (phospho S386) antibody [EPR2346] (ab76493)

      Dot blot analysis of IRF3 single phospho peptide pS386 (lane 1) and IRF3 non-phospho peptide (lane 2) with unpurified ab76493 at 1/1000. Blocking and diluting buffer was 5% NFDM/TBST. The secondary antibody used was ab97051 Peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.

    References

    This product has been referenced in:

    • Caller LG  et al. Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G2 Arrest and Highly Effective Evasion of Innate Immune Sensing. J Virol 93:N/A (2019). Read more (PubMed: 31142673) »
    • He X  et al. RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation. EMBO J 38:e100978 (2019). Read more (PubMed: 31304625) »
    See all 67 Publications for this product

    Publishing research using ab76493? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-6 of 6 Abreviews or Q&A

    Western blot abreview for Anti-IRF3 (phospho S386) antibody [EPR2346]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    25 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Mouse Cell lysate - whole cell (liver)
    Specification
    liver
    Treatment
    20 ug/ml poly(I:C)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 26 2014

    Question

    Hi, can you recommend an isotype control to Ab below that would be suitable for flow cytometry?

    https://www.abcam.com/irf3-phospho-s386-antibody-epr2346-ab76493.html

    Read More
    Answer

    Thank you for contacting us. The rabbit monoclonal isotyoe control ab128142 should be suitable for your purposes in Flow Cytometry as well. Do you also need any secondary antibodies for your experiment? We offer many fluorophore conjugated secondaries as well.

    Read More

    Question

    Zum oberen Bild:
    Geblockt wurde mit 5 % BSA in PBST für 1h bei RT. Anschließend wurde der P-IRF3 AK von Abcam (1:500 in 5 % BSA/PBST) zugegeben und über Nacht inkubiert. Das Ergebnis zeigt den P-IRF3 Antikörper. Es wurde kein anderer Antikörper zuerst detektiert.

    Zum unteren Bild:
    Blocking: 5 % Milchpulver in PBST, 1h, RT
    1. Antikörper: P-IRF3 von Abcam (1:1000 in 5% BSA/PBST), über Nacht
    Auch in diesem Fall hat keine andere Detektion vorher stattgefunden.
    Darunter ist die anschließende ß-Aktin Detektion gezeigt.
    2. Stellt das oberes Bild Actin einen Blot mit Actin dar? Wenn ja, welcher Antikörper wurde zuerst inkubiert, der Actin oder phospho-IRF3?
    Vielen Dank für Ihre Kooperation.
    Das obere Bild stellt die P-IRF3 Detektion dar. P-IRF3 wurde immer zuerst detektiert, falls weitere Antikörper später noch detektiert werden sollten.
    Die Ladungskontrolle (Aktin) wurde als stärkstes Signal immer zum Schluss detektiert.

    Noch kurz zur Info: neben dem jeweiligen Bild ist immer der Name des verwendeten Antiköpers geschrieben.

    Read More
    Answer

    Vielen Dank für Ihre Antwort.

    Ich möchte Ihnen einen kostenlosen Ersatz oder eine Gutschrift anbieten, da der Antikörper nicht so funktioniert wie wir diesen auf unserem Datenblatt beschreiben. Dazu benötige ich allerdings das ungefähre Bestelldatum, bzw. die Bestellreferenznummer.
    Noch einmal vielen Dank für Ihre Kooperation.

    Read More

    Question

    Hallo,
    attatched you will finde some excamples of the western blots.
    1) Abcam product code ab
    Ab-76493
    2) Abcam order reference number or product batch number
    lot: GR15710-3
    3) Description of the problem
    No P-IRF3 detection in any ways
    4) Sample preparation:
    Type of sample (whole cell lysates, fraction, recombinant protein…):
    Lysis buffer:
    RIPA ([50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0,5 % (vol/vol) Igepal, 0,25 % (vol/vol) Dexychloric acid] supplemented with phosphatase inhibitor and protease inhibitor
    Protease inhibitors:
    Protease inhibitor (Roche, 11836170001 ) (1 tablet in 10 ml RIPA)
    Phosphatase inhibitors
    1% (vol/vol) Phosphatase inhibitor (Thermo Scientific, 78420)
    Reducing agent
    5% (vol/vol) ß-Mercaptoethanol, Sigma

    Boiling for ≥5 min? yes
    Protein loaded ug/lane:
    40 µg
    Positive control:
    Poly I:C treated human hepatocytes
    Or
    LPS treated human hepatocytes
    Or
    LPS treated, time course, human hepatocytes
    Negative control
    Untreated human hepatocytes
    5) Percentage of gel
    Tested in:
    4-15 % TGX Precast Gels (BioRad)
    Any kD TGX Precast Gels (BioRad)
    4-12 % Bis-Tris Gel (Invitrogen)
    Type of membrane
    Tested:
    PVDF
    Nitrocellulose
    Protein transfer verified
    Ponseau S and by eye (marker)

    Blocking agent and concentration
    Tested:
    5 % BSA/PBST
    5 % Milkpowder/PBST
    Blocking time
    1h
    Blocking temperature
    RT
    6) Primary antibody (If more than one was used, describe in “additional notes”) :
    Concentration or dilution
    Tested:
    1:1000
    1:200
    1:500
    Diluent buffer
    5 % BSA/PBST
    5 % Milkpowder/PBST
    Incubation time, Incubation temperature:
    Tested:
    1 h, RT
    Over night, 4°C
    7) Secondary antibody:
    Species: goat
    Reacts against: rabbit
    Concentration or dilution: tested 1: 2500, 1:5000
    Diluent buffer : 5 % BSA/PBST
    Incubation time: 1h
    Incubation temperature: 4°C
    Fluorochrome or enzyme conjugate: HRP
    8) Washing after primary and secondary antibodies:
    Buffer: PBST
    Number of washes 3x 5 or 2x 10 min
    9)Detection method: Chemoluminescence
    10) How many times have you run this staining? ˜8 times
    Do you obtain the same results every time? yes
    What steps have you altered to try and optimize the use of this antibody?
    - Blockings
    - Gels
    - Concentrations
    - Incubationtime
    - Membrane
    - Cells

    Read More
    Answer

    Vielen Dank für das Ausfüllen unseres Fragebogens.

    Ich habe nur noch zwei Fragen bezüglich Ihres Protokolls:


    1.Haben Sie einen Blot nur mit BSA geblockt und anschliessend den prim. Antikörper in TBST+ BSA inkubiert?

    2. Stellt das oberes Bild Actin einen Blot mit Actin dar? Wenn ja, welcher Antikörper wurde zuerst inkubiert, der Actin oder phospho-IRF3?

    Vielen Dank für Ihre Kooperation.

    Read More

    Question

    Inquiry: Dear Sir or Madam, I have bought the Phospho-IRF3 antibody. I´m working with human hepatocytes and in my experimental setup I included positive controls (LPS or Poly I:C treated human hepatocytes). It also didn´t work with human stellate cells, kupffer cells and liver sinusoidal endothelial cells. I usually load 40 to 50 µg protein. I never had success to detect phospho IRF3. I also tested different conditions like Blocking (5% BSA or 5 % Milk in PBST or TBST). Could it be a problem of the antibody batch (lot: GR15710-3). Would it be possible to get a test-aliquot again maybe with an additional positive control? I look forward to your reply.

    Read More
    Answer

    Thank you for contacting us.

    I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. We have not received any other negative feedback on this batch or any other batch during the last year.

    I would like however to investigate the problem you are experiencing. Iwould like therefore to gather some more information about the protocol you have been using and am attaching therefore questionnaire. Please take 5 minutes to fill it out. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results.This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

    I look forward to receiving your reply.

    Read More

    Question

    Can you please explain why the ICC image shows positive staining in untreated MCF7 cells? The WB image shows negative results in these untreated cells.

    Read More
    Answer

    Thank you for your call yesterday and for your patience while I have been in touch with the lab regarding ab76493. The phospho-specificity of the antibody was determined by Western blot and dot blot. The fluorescent detection may be more sensitive than the WB detection system, so it is possible that ICC will show different results than in Western blot. However, since the specificity was never determined in ICC it is possible that some non-specific binding contributes to the staining. The ICC results were not further investigated, so unfortunately we don't have a definite explanation for these results. You can compare treated and untreated cells to each other, and we would expect there to be a noticeable difference in the signal between these groups. If you have any trouble with this antibody in ICC, please let me know and I will be happy to help you further. If you have any further questions, don't hesitate to contact me.

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