NFkB p65 Transcription Factor Assay Kit (Chemiluminescent) (ab207221) is a high throughput assay to quantify NFkB p65 activation in nuclear extracts.
Luminescent
Nuclear Extracts
Semi-quantitative
3h 30m
< 400 ng/well
NFkB p65 Transcription Factor Assay Kit (Chemiluminescent) (ab207221) is a high throughput assay to quantify NFkB p65 activation in nuclear extracts.
Luminescent
Nuclear Extracts
Semi-quantitative
3h 30m
Microplate reader
< 400 ng/well
Dry Ice
Multi
Multi
Please refer to protocols
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:
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.
NF-kB p65 also known as RelA is a significant component of the NF-kB protein complex. This complex usually involves a molecular weight for p65 of approximately 65 kDa. NF-kB p65 is mechanistically a transcription factor that regulates genes involved in inflammation cell survival and immune response. Expression of p65 is widely seen in various cell types including immune cells and epithelial cells suggesting its role in numerous physiological processes.
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.
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.
NF-kB p65 is implicated in conditions such as cancer and autoimmune diseases. Its dysregulation can lead to persistent activation of inflammatory pathways contributing to tumorigenesis and chronic inflammation. In the context of these diseases proteins such as the p50 subunit and the IKK complex are often involved reflecting the importance of tight regulation of NF-kB signaling in preventing pathological conditions.
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