Overview

  • Product name

    IRF3 Transcription Factor Assay Kit (Colorimetric)
  • Detection method

    Colorimetric
  • Sample type

    Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Sensitivity

    500 ng/well
  • Assay time

    3h 30m
  • Species reactivity

    Reacts with: Human
  • Product overview

    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.

  • Notes

    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.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please 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
  • Research areas

  • Function

    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.
  • Tissue specificity

    Expressed constitutively in a variety of tissues.
  • Sequence similarities

    Belongs to the IRF family.
    Contains 1 IRF tryptophan pentad repeat DNA-binding domain.
  • Post-translational
    modifications

    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.
  • Cellular localization

    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
  • Alternative names

    • IIAE7
    • Interferon regulatory factor 3
    • IRF 3
    • IRF-3
    • IRF3
    • IRF3_HUMAN
    • MGC94729
    see all
  • Database links

Images

  • Different amounts of poly (I/C) treated COS7 cells were tested for IRF3 activation. These results are provided for demonstration purposes only.

Protocols

References

ab207210 has not yet been referenced specifically in any publications.

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