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Anti-IRF3 antibody [EPR2418Y] ab68481 is a rabbit monoclonal antibody that is used in IRF3 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human and mouse samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EPR2418Y has been tried and trusted by researchers since 2008 and is cited in >90 publications
- Specificity confirmed with IRF3 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation

New 20 ul size available


Images

Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481), expandable thumbnail
  • Immunocytochemistry/ Immunofluorescence - Anti-IRF3 antibody [EPR2418Y] (AB68481), expandable thumbnail
  • Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (AB68481), expandable thumbnail
  • Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481), expandable thumbnail
  • Western blot - Anti-IRF3 antibody [EPR2418Y] (AB68481), expandable thumbnail

Publications

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IPWBICC/IFFlow Cyt (Intra)IHC-P
Human
Not recommended
Tested
Tested
Tested
Tested
Mouse
Not recommended
Tested
Expected
Expected
Tested

Not recommended
Not recommended

Species

Mouse

Dilution info

-

Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species

Human

Dilution info

-

Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species

Mouse

Dilution info

1/1000

Notes

-

Species

Human

Dilution info

1/1000

Notes

-

Tested
Tested

Species

Human

Dilution info

1/100

Notes

-

Expected
Expected

Species

Mouse

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

1/160

Notes

Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. MeOH fixationis recommended.

Expected
Expected

Species

Mouse

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/500

Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Species

Human

Dilution info

1/500

Notes

-

Associated Products

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Target data

Function

Key transcriptional regulator of type I interferon (IFN)-dependent immune responses which plays a critical role in the innate immune response against DNA and RNA viruses (PubMed:22394562, PubMed:25636800, PubMed:27302953). Regulates the transcription of type I IFN genes (IFN-alpha and IFN-beta) and IFN-stimulated genes (ISG) by binding to an interferon-stimulated response element (ISRE) in their promoters (PubMed:11846977, PubMed:16846591, PubMed:16979567, PubMed:20049431, PubMed:32972995). Acts as a more potent activator of the IFN-beta (IFNB) gene than the IFN-alpha (IFNA) gene and plays a critical role in both the early and late phases of the IFNA/B gene induction (PubMed:16846591, PubMed:16979567, PubMed:20049431). Found in an inactive form in the cytoplasm of uninfected cells and following viral infection, double-stranded RNA (dsRNA), or toll-like receptor (TLR) signaling, is phosphorylated by IKBKE and TBK1 kinases (PubMed:22394562, PubMed:25636800, PubMed:27302953). This induces a conformational change, leading to its dimerization and nuclear localization and association with CREB binding protein (CREBBP) to form dsRNA-activated factor 1 (DRAF1), a complex which activates the transcription of the type I IFN and ISG genes (PubMed:16154084, PubMed:27302953, PubMed:33440148). Can activate distinct gene expression programs in macrophages and can induce significant apoptosis in primary macrophages (PubMed:16846591). In response to Sendai virus infection, is recruited by TOMM70:HSP90AA1 to mitochondrion and forms an apoptosis complex TOMM70:HSP90AA1:IRF3:BAX inducing apoptosis (PubMed:25609812). Key transcription factor regulating the IFN response during SARS-CoV-2 infection (PubMed:33440148).

Alternative names

Interferon regulatory factor 3, IRF-3, IRF3

Recommended products

Anti-IRF3 antibody [EPR2418Y] ab68481 is a rabbit monoclonal antibody that is used in IRF3 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human and mouse samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EPR2418Y has been tried and trusted by researchers since 2008 and is cited in >90 publications
- Specificity confirmed with IRF3 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation

New 20 ul size available

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EPR2418Y

Purification technique

Affinity purification Protein A

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

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 monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

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

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

IRF3 also known as Interferon Regulatory Factor 3 acts as an important transcription factor in the immune response. It has a molecular weight of approximately 47 kDa. The IRF3 protein is mainly expressed in the cytoplasm and nucleus of various cell types including immune cells such as macrophages and dendritic cells. The protein becomes activated through phosphorylation a process frequently identified in its phosphorylated form phospo-IRF3 or p-IRF3 which facilitates its role in immune function.

Biological function summary

IRF3 participates in the regulation of type I interferon (IFN) response a fundamental antiviral defense mechanism. IRF3 when phosphorylated forms a complex with CBP/p300 which then translocates to the nucleus to drive the expression of IFN-stimulated genes. This action strengthens the innate immune response and boosts the body's ability to counteract viral infections. Its activity and regulation are significant for maintaining a balanced immune response without excessive inflammation.

Pathways

IRF3 is involved in the Toll-like receptor (TLR) and RIG-I-like receptor (RLR) signaling pathways both essential in pathogen recognition and response. Within these pathways IRF3 interacts with proteins such as MAVS and TBK1 to propagate immune signaling. The activation of IRF3 in these pathways results in the production of type I interferons and other cytokines orchestrating an effective antiviral response. These interactions highlight the protein's central role in mediating immune signaling cascades.

Associated diseases and disorders

IRF3's malfunction or deregulation can contribute to autoimmune diseases and antiviral deficiencies. Conditions such as systemic lupus erythematosus (SLE) and chronic hepatitis B infection are linked to IRF3 activity. During autoimmune responses or viral persistence the aberrant activation of IRF3 can lead to inappropriate immune responses. The connection with proteins like STAT1 in these conditions highlights the complex network IRF3 engages in facilitating its impact on disease progression and immune dysregulation.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

16 product images

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Lanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.

    ab68481 was shown to react with IRF3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human IRF3 knockout HeLa cell line ab255345 (knockout cell lysate Human IRF3 knockout HeLa cell lysate ab263784) was used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.

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

    Lane 1: Jurkat cell lysate at 20 µg

    Lane 2: MCF7 cell lysate at 20 µg

    Lane 3: Wild-type HeLa cell lysate at 20 µg

    Lane 4: IRF3 knockout HeLa cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

    Performed under reducing conditions.

    Predicted band size: 47 kDa

    Observed band size: 37 kDa, 51 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-IRF3 antibody [EPR2418Y] (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) (Goat Anti-Rabbit IgG H&L (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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    2. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

  • Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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 MeOHinstead of PFA toget optimal results.

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab68481 was shown to react with IRF3 in wild-type A549 cells in western blot with loss of signal observed in IRF3 knockout cell line Human IRF3 knockout A549 cell line ab267098 (IRF3 knockout cell lysate Human IRF3 knockout A549 cell lysate ab256954). Wild-type and IRF3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab68481 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (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.

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

    Lane 1: Wild-type A549 cell lysate at 20 µg

    Lane 2: IRF3 knockout A549 cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 47 kDa

    Observed band size: 50 kDa

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Lanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.

    ab68481 was shown to react with IRF3 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when IRF3 knockout samples were examined. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were both diluted to 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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: Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution

    Lane 1: Wild-type HAP1 cell lysate at 20 µg

    Lane 2: IRF3 knockout HAP1 cell lysate at 20 µg

    Lane 3: HeLa cell lysate at 20 µg

    Lane 4: Jurkat cell lysate at 20 µg

    Predicted band size: 47 kDa

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and Dilution buffer: 5% NFDM/TBST

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

    Lane 1: Human fetal heart lysate at 10 µg

    Lane 2: Human fetal kidney lysate at 10 µg

    Secondary

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

    Predicted band size: 47 kDa

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab68481 was shown to react with IRF3 in wild-type A549 cells in western blot with loss of signal observed in IRF3 knockout cell line Human IRF3 knockout A549 cell line ab267097 (IRF3 knockout cell lysate Human IRF3 knockout A549 cell lysate ab256953). Wild-type and IRF3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab68481 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (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.

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

    Lane 1: Wild-type A549 cell lysate at 20 µg

    Lane 2: IRF3 knockout A549 cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 47 kDa

    Observed band size: 50 kDa

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and Dilution buffer: 5% NFDM/TBST

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

    Lane 1: THP-1 (Human monocytic leukemia cells) whole cell lysates at 10 µg

    Lane 2: HepG2 (Human liver hepatocellular carcinoma) whole cell lysates at 10 µg

    Lane 3: Daudi (Human Burkitt's lymphoma cell line) whole cell lysates at 10 µg

    Secondary

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

    Predicted band size: 47 kDa

    Observed band size: 51 kDa

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and Dilution buffer: 5% NFDM/TBST

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

    Lane 1: HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 20 µg

    Lane 2: Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

    Secondary

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

    Predicted band size: 47 kDa

    Observed band size: 51 kDa

  • Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Intracellular 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).

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and Dilution buffer: 5% NFDM/TBST

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

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

    Lane 1: Mouse heart lysate at 10 µg

    Lane 2: Mouse spleen lysate at 10 µg

    Lane 3: NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates at 10 µg

    Secondary

    All lanes: 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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF3 antibody [EPR2418Y] (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.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The identity of the bands between 25 kDa and 35 kDa are unknown.

    In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

    In Western blot, Anti-IRF3 antibody [EPR2418Y] (ab68481) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-IRF3 (phospho S396) antibody [EPR28686-189] (Anti-IRF3 (phospho S396) antibody [EPR28686-189] ab320082) at 1/1000 dilution

    Lane 1: Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 60 µg

    Lane 2: THP-1 treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate at 60 µg

    Secondary

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

    Observed band size: 60 kDa, 36 kDa

    Exposure time: 48s

  • Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481), expandable thumbnail

    Western blot - Anti-IRF3 antibody [EPR2418Y] (ab68481)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-IRF3 (phospho S396) antibody [EPR28686-189] ab320082 was shown to bind specifically to IRF3. Target of interest was observed at 60 kDa in wild-type Hela cell lysates (lane 2) with no signal observed at this size in IRF3 knockout cell line (lane 4) (lane 4, knockout cell line Human IRF3 knockout HeLa cell line ab255345 / knockout cell lysate Human IRF3 knockout HeLa cell lysate ab263784).

    Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

    In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

    In Western blot, Anti-IRF3 antibody [EPR2418Y] (ab68481) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-IRF3 (phospho S396) antibody [EPR28686-189] (Anti-IRF3 (phospho S396) antibody [EPR28686-189] ab320082) at 1/1000 dilution

    Lane 1: Untreated wild-typeHeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 60 µg

    Lane 2: Wild-type HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (untreated membrane) at 60 µg

    Lane 3: Untreated IRF3 knockout HeLa whole cell lysate (untreated membrane) at 60 µg

    Lane 4: IRF3 knockout HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (untreated membrane) at 60 µg

    Lane 5: Untreated wild-typeHeLa whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

    Lane 6: Wild-type HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

    Lane 7: Untreated IRF3 knockout HeLa whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

    Lane 8: IRF3 knockout HeLa treated first with 80nM TPA for 24h, then change fresh medium, transfect 10ug/ml poly(dA:dT) for 2h whole cell lysate (alkaline phosphatase treated membrane) at 60 µg

    Secondary

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

    Performed under reducing conditions.

    Observed band size: 60 kDa, 36 kDa

    Exposure time: 92s

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For this product, it's our understanding that no specific protocols are required. You can:

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