Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
- Sensitivity: 500 ng/well
Product nameIRF3 Transcription Factor Assay Kit (Colorimetric)
See all IRF3 kits
Sample typeNuclear Extracts
Assay time3h 30m
Species reactivityReacts with: Human
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.
Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 5 x 96 tests 10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml 10X Wash Buffer 1 x 22ml 1 x 110ml 96-well IRF-3 assay plate 1 unit 5 units Anti-mouse HRP-conjugated IgG 1 x 10µl 1 x 50µl Binding Buffer 1 x 10ml 1 x 50ml Cos-7 nuclear extract (Poly (I-C) 2 hr) (2.5 µg/µL) 1 x 40µl 1 x 200µl Developing Solution 1 x 11ml 1 x 55ml Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl IRF-3 antibody (human) 1 x 10µl 1 x 25µl Lysis Buffer 1 x 10ml 1 x 50ml Mutated oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl Plate sealer 1 unit 5 units Poly [d(l-c)] (17 µg/μL) 1 x 100µl 1 x 500µl Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl Stop Solution 1 x 11ml 1 x 55ml STAT Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
FunctionMediates 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 specificityExpressed constitutively in a variety of tissues.
Sequence similaritiesBelongs to the IRF family.
Contains 1 IRF tryptophan pentad repeat DNA-binding domain.
modificationsConstitutively 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 localizationCytoplasm. 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
- Interferon regulatory factor 3
- IRF 3
ab207210 has been referenced in 1 publication.
- Ambrose RL et al. Molecular characterisation of ILRUN, a novel inhibitor of proinflammatory and antimicrobial cytokines. Heliyon 6:e04115 (2020). PubMed: 32518853