Description
Key facts
Detection method:Colorimetric,
Sample types:Nuclear Extracts,
Reacts with:Human,
Assay type:Semi-quantitative,
Sensitivity:= 500 ng/well,
Assay time:3h 30m,
Assay Platform:Microplate reader
Product details:
IRF3 Transcription Factor Assay Kit (Colorimetric) (ab207210) is a high throughput assay to quantify IRF3 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.
A specific double stranded DNA sequence containing the IRF3 consensus binding site (5' – GAAACTGAAACT – 3') has been immobilized onto a 96-well plate. Active IRF3 present in the nuclear extract specifically binds to the oligonucleotide. IRF3 is detected by a primary antibody that recognizes an epitope of IRF3 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout at OD 450 nm. This product detects only human IRF3.
Key performance and benefits:
Assay time: 3.5 hours (cell extracts preparation not included).
Detection limit: < 0.5 μg nuclear extract/well.
Detection range: 0.5 – 10 μg nuclear extract/well.
The interferon (IFN) regulatory factor (IRF) family is a group of transcription factors that have extensive homology in their DNA-binding domain (DBD). The many members of the IRF family are involved in the regulation of interferon (IFN) a and b and play a role in host anti-viral immune regulation, cell growth and hematopoietic development. The N-terminal binding domain of IRFs, the distinct feature of the family, is a modified helix-turn-helix characterized by repeated tryptophan residues separated by 10 to 18 amino acids. All IRFs, except IRF1 and IRF2, have an IRF association domain (IAD) that is responsible for the interaction with other family members or with transcription factors such as PU.1, E47 and STAT. Another association domain (IAD2), present only in IRF1 and IRF2, is important for interaction with IRF8. A nuclear localization signal has been identified in IRF1, and similar sequences may also be present in other family members. A bipartite nuclear retention signal located within the N-terminus of the DBD has been identified in IRF4, IRF8 and IRF9. IRFs also possess a transactivation domain in the middle of the protein.
IRFs bind DNA as dimers on sequences such as the IFN-stimulated response element (ISRE), AGTTTCNNCNY, the IFN consensus sequence (ICS), R(G/C)TTTC, or the IFN-regulatory factor element (IRFE), G(A)AAA(G/C)YGAAA(G/C)Y. While IRF3 is constitutively expressed, the expression of other IRFs is induced by such stimuli as type I and II IFN, double-stranded RNA or the presence of viral components, which can also induce the activity of the IRF factors after they have been synthesized. IRF factors can cooperate with other factors with neighboring binding sites on promoters. For example, the IFN-b promoter provides the stepping stone for the formation of an "enhanceosome" containing ATF-2, c-Jun, IRF3, NFkB and CBP.
In mammals, ten IRF family members have been identified. IRF1, an activator, is involved in mature lymphocyte apoptosis after DNA-damage. IRF2 is mainly a repressor, but can also play a role in histone H4 expression. Both IRF1 and IRF2 interact with the co-activator P/CAF. IRF1 has been described as a tumor suppressor and IRF2 as a proto-oncogene. IRF3 is a component of DRAF, a complex containing the acetylase CBP/p300, which binds ISRE-like sequences. Double-stranded RNA (dsRNA) or poly (I-C), a synthetic form of dsRNA, elicits IRF3 activation. IRF4 is required for the function and homeostasis of B and T-cells and can also interact with the Ets-family member PU.1 and with the E47 form of E2A. IRF7 helps induce the IFN-a gene and repress the EBNA-1 gene from the Epstein-Barr virus. The expression of IRF7 is induced by type I IFN, which is important for amplification of the signaling pathway. IRF8 (ICSBP) can interact with IRF1 and 2, but primarily acts as a repressor. IRF9 (ISGF3g/p48) is part of the ISGF3 transcription factor, together with STAT1 and STAT2. IRF10 functions in the late stages of anti-viral defense by regulating IFNg target genes.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
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.
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.
