重组Anti-IRF3抗体[EPR2418Y] (ab68481)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR2418Y] to IRF3
- Suitable for: Flow Cyt (Intra), ICC/IF, WB, IHC-P
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
概述
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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 -
宿主
Rabbit -
经测试应用
适用于: Flow Cyt (Intra), ICC/IF, WB, IHC-Pmore details
不适用于: IP -
种属反应性
与反应: Mouse, Human -
免疫原
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阳性对照
- WB: A549, 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, HAP1-wt cells.
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常规说明
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.
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol, 0.05% BSA, 59% PBS -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
EPR2418Y -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Assay kits
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Compatible Secondaries
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Conjugation kits
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Immunizing Peptide (Blocking)
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Isotype control
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KO cell lines
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KO cell lysates
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Recombinant Protein
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab68481于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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Flow Cyt (Intra) |
1/160.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. MeOH fixationis recommended. |
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ICC/IF |
1/100.
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WB | (1) |
1/1000. Detects a band of approximately 51 kDa (predicted molecular weight: 47 kDa).Can be blocked with IRF3 peptide (ab203561).
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IHC-P |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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说明 |
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Flow Cyt (Intra)
1/160. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. MeOH fixationis recommended. |
ICC/IF
1/100. |
WB
1/1000. Detects a band of approximately 51 kDa (predicted molecular weight: 47 kDa).Can be blocked with IRF3 peptide (ab203561). |
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
靶标
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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
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数据库链接
- Entrez Gene: 3661 Human
- Entrez Gene: 54131 Mouse
- Omim: 603734 Human
- SwissProt: Q14653 Human
- SwissProt: P70671 Mouse
- Unigene: 289052 Human
- Unigene: 75254 Human
- Unigene: 3960 Mouse
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别名
- IIAE7 antibody
- Interferon regulatory factor 3 antibody
- IRF 3 antibody
see all
图片
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All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution
Lane 1 : Jurkat cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : IRF3 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDaLanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - 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 ab255345 (knockout cell lysate ab263784) was used. Wild-type and IRF3 knockout samples were subjected to SDS-PAGE. ab68481 and 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 (ab216773) and 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.
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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. -
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 (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (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.
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All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : IRF3 knockout A549 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - 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 ab267098 (IRF3 knockout 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : IRF3 knockout A549 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - 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 ab267097 (IRF3 knockout 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-IRF3 antibody [EPR2418Y] (ab68481) at 1/1000 dilution
Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : IRF3 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 47 kDaLanes 1 - 4: Merged signal (red and green). Green - ab68481 observed at 50 kDa. Red - 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 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 (ab216773) and 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 : 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
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution
Predicted band size: 47 kDaBlocking and Dilution buffer: 5% NFDM/TBST
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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
All lanes : 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
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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
All lanes : 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
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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
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 kDaBlocking and Dilution buffer: 5% NFDM/TBST
The slightly smaller molecular mass observed in mouse than in human is supported by literature.
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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).
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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.
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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.
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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.
实验方案
数据表及文件
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SDS download
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Datasheet download
文献 (57)
ab68481 被引用在 57 文献中.
- Zhao M et al. The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA. EMBO Rep 23:e53166 (2022). PubMed: 34779554
- Huang YH et al. Heat Shock Protein 60 Restricts Release of Mitochondrial dsRNA to Suppress Hepatic Inflammation and Ameliorate Non-Alcoholic Fatty Liver Disease in Mice. Int J Mol Sci 23:N/A (2022). PubMed: 35009003
- Chen Q et al. Truncated PARP1 mediates ADP-ribosylation of RNA polymerase III for apoptosis. Cell Discov 8:3 (2022). PubMed: 35039483
- Ma C et al. p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling. PLoS Pathog 18:e1010295 (2022). PubMed: 35180274
- Chen L et al. Oncolytic Zika virus promotes intratumoral T cell infiltration and improves immunotherapy efficacy in glioblastoma. Mol Ther Oncolytics 24:522-534 (2022). PubMed: 35229030