NFkB p65 Transcription Factor Assay Kit (Chemiluminescent)
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NFkB p65 Transcription Factor Assay Kit (Chemiluminescent) (ab207221) is a high throughput assay to quantify NFkB p65 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 NFkB p65 consensus binding site (5' – GGGACTTTCC – 3') has been immobilized onto a 96-well plate. Active NFkB p65 present in nuclear specifically binds to the oligonucleotide. NFkB p65 is detected by a primary antibody that recognizes an epitope of NFkB p65 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides a sensitive chemiluminescent readout that can be quantified using a luminometer or CCD camera system. This product detects human, mouse and rat NFkB p65.
Key performance and benefits:
- Assay time: 3.5 hours (cell extracts preparation not included).
- Detection limit: < 40 ng nuclear extract/well.
- Detection range: 0.039 – 2.5 μg nuclear cell extract/well.
The transcription factor NFkB is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses. NFkB is composed of homo- and heterodimeric complexes of members of the Rel (NFkB) family. There are five subunits of the NFkB family in mammals: p50, p65 (RelA), c-Rel, p52 and RelB. These proteins share a conserved 300 amino acid sequence in the N-terminal region, known as the Rel homology domain, that mediates DNA binding, protein dimerization and nuclear localization. Various dimer combinations of the NFkB subunits have distinct DNA binding specificities and may serve to activate specific sets of genes such as adhesion molecules, immunoreceptors and cytokines. The p50/p65 heterodimers are some of the most common dimers found in the NFkB signaling pathway. RelA (p65) heterodimers with both p50 and p52 participate in target gene transactivation.
In the majority of cells, NFkB exists in an inactive form in the cytoplasm, bound to the inhibitory IkB proteins. Treatment of cells with various inducers results in the phosphorylation, ubiquitination and subsequent degradation of IkB proteins (For studying the phosphorylation state of IkBa. Proteolytic cleavage of p105 results in two proteins: p50, which has DNA-binding activity but no transactivation domain, and its antagonist, the inhibitory IkBg protein. This results in the release of NFkB dimers, which subsequently translocate to the nucleus, where they activate appropriate target genes. NFkB can be activated by a number of stimuli, including components of bacterial cell walls, such as lipopolysaccharide, or inflammatory cytokines, such as TNF-α or IL-1β.
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Biological function summary
NF-kB p65 acts as part of the larger NF-kB complex usually forming a heterodimer with other family members like p50. This complex translocates to the nucleus upon activation where it binds specific DNA sequences to regulate gene expression. The p65 subunit is essential for transactivating target genes involved in immune and inflammatory responses. Its regulation is important for proper cellular functioning especially in the maintenance of immune homeostasis and inflammatory response.
Pathways
NF-kB p65 participates in critical pathways like the NF-kB signaling and Toll-like receptor pathways. These pathways are fundamental for initiating immune responses and contribute to the regulation of apoptosis and cellular stress responses. In the context of these pathways the IKK complex plays a pivotal role in NF-kB activation leading to the phosphorylation and subsequent degradation of inhibitors that retain NF-kB p65 in the cytoplasm therefore allowing its movement to the nucleus.
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Publications (1)
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Cell reports 25:561-570.e6 PubMed30332638
2018
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