IRF3 Transcription Factor Assay Kit (Colorimetric)

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.