Recombinant Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR24260-55] to Interferon regulatory factor 9/IRF-9
- Suitable for: WB, IP, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55]
See all Interferon regulatory factor 9/IRF-9 primary antibodies -
Description
Rabbit monoclonal [EPR24260-55] to Interferon regulatory factor 9/IRF-9 -
Host species
Rabbit -
Tested applications
Suitable for: WB, IP, IHC-P, ICC/IFmore details
Unsuitable for: Flow Cyt (Intra) -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa cells treated with IFN alpha (10 ng/ml), THP-1 cells, Human ovary cancer, Human liver cancer lysates. IHC-P: HeLa cells treated with IFN alpha (10 ng/ml), Human spleen and Human lung, Clear carcinoma of human kidney tissues. ICC/IF: HeLa cells treated with IFN alpha-1 (10 ng/ml). IP: HeLa cells treated with IFN alpha-1 (10 ng/ml), THP-1 cells.
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General notes
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 production
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
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR24260-55 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab271043 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 |
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WB |
1/1000. Predicted molecular weight: 44 kDa.
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IP |
1/30.
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IHC-P |
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
1/50.
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Notes |
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WB
1/1000. Predicted molecular weight: 44 kDa. |
IP
1/30. |
IHC-P
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/50. |
Target
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Function
Transcription regulatory factor that mediates signaling by type I IFNs (IFN-alpha and IFN-beta). Following type I IFN binding to cell surface receptors, Jak kinases (TYK2 and JAK1) are activated, leading to tyrosine phosphorylation of STAT1 and STAT2. The phosphorylated STATs dimerize, associate with IRF9/ISGF3G to form a complex termed ISGF3 transcription factor, that enters the nucleus. ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of interferon stimulated genes, which drive the cell in an antiviral state. -
Sequence similarities
Belongs to the IRF family.
Contains 1 IRF tryptophan pentad repeat DNA-binding domain. -
Cellular localization
Cytoplasm. Nucleus. Translocated into the nucleus upon activation by IFN-alpha/beta. - Information by UniProt
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Database links
- Entrez Gene: 10379 Human
- Omim: 147574 Human
- SwissProt: Q00978 Human
- Unigene: 1706 Human
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Alternative names
- IFN alpha responsive transcription factor subunit antibody
- IFN-alpha-responsive transcription factor subunit antibody
- Interferon regulatory factor 9 antibody
see all
Images
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All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution
Lane 1 : wild-type HeLa Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate
Lane 2 : IRF9 knockout HeLa treated: hIFN-a1 (10 ng/ml, 16 h) cell lysate
Lane 3 : wild-type HeLa Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate
Lane 4 : IRF9 knockout HeLa vehicle control: hIFN-a1 (0 ng/ml, 16 h) cell lysate
Lane 5 : THP-1 cell lysate
Lane 6 : ACHN cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab266051 (CRISPR-Cas9 edited cell lysate ab258472). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution
Lane 1 : wild-type A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate
Lane 2 : IRF9 knockout A549 Treated Interferon-alpha1 (hIFN-a1) (10 ng/ml, 16 h) cell lysate
Lane 3 : wild-type A549 Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate
Lane 4 : IRF9 knockout A549 Control Interferon-alpha1 (hIFN-a1) (0 ng/ml, 16 h) cell lysate
Lane 5 : THP-1 cell lysate
Lane 6 : ACHN cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab267119 (CRISPR-Cas9 edited cell lysate ab258473). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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ab271043 staining Interferon regulatory factor 9 in wild-type A549 cells and IRF9 knockout A549 cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
ab271043 staining Interferon regulatory factor 9 in wild-type HeLa cells and IRF9 knockout HeLa cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution
Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa treated with 10ng/ml human IFN alpha 1 (ab48750) for 16 hours, whole cell lysate
Lane 3 : THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lysates/proteins at 40 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 44 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
IRF9 expression is increased by IFN-alpha (PMID:18190617).
Exposure time: 15 seconds.
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All lanes : Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] (ab271043) at 1/1000 dilution
Lane 1 : Human ovary cancer tissue lysate
Lane 2 : Human liver cancer tissue lysate
Lysates/proteins at 40 µg per lane.
Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution (VeriBlot for IP secondary antibody(HRP))
Predicted band size: 44 kDa
Observed band size: 30,48 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
IFR9 can be degraded by the EV71-encoded 3C protease. A 48-kDa full length and 30-kDa cleaved IRF9 are observed. The molecular weight is consistent with what have been described in literature (PMID:21536800).
Exposure time: 3 minutes.
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Immunohistochemical analysis of paraffin-embedded HeLa (human cervix adenocarcinoma epithelial cell) cells (A) and HeLa cells treated by 10 ng/ml IFN for 18h (B) tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Almost no staining on HeLa cells (A) and strongly positive staining on HeLa cells treated by 10 ng/ml IFN for 18h (B). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use (Bond™ Polymer Refine Detection). Positive staining on human spleen. The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Human lung tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung (PMID: 30355451). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Clear cell carcinoma of human kidney tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Clear cell carcinoma of human kidney (PMID: 30355451). The section was incubated with ab271043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/50 dilution(9.3 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/500 dilution (Green). Confocal image showing increased nuclear and weak cytoplasmic staining in HeLa cells treated with Interferon alpha-1 (10 ng/ml) for 16 h is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/500 dilution.
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Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg treated HeLa whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours, whole cell lysate 10 ug
Lane 2: ab271043 IP in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271043 in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
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Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: THP-1 whole cell lysate 10 ug
Lane 2: ab271043 IP in THP-1 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271043 in THP-1 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab271043 has not yet been referenced specifically in any publications.