Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Pre-coated microplate (12 x 8 well strips)
- Sample type: Cell Lysate, Nuclear Extracts
Product nameNF-kappaB RelA/p65 Transcription Factor Activity Assay Kit (Colorimetric)
See all NF-kB p65 kits
Sample typeCell Lysate, Nuclear Extracts
Assay durationMultiple steps standard assay
Species reactivityReacts with: Human
NF-kappaB RelA/p65 Transcription Factor Activity Assay (Colorimetric) (ab289845, K2093) is a 96-well plate based colorimetric assay to measure the activation of human NF-kappaB p65 in nuclear extracts or cell lysates. The kit offers easy, rapid, sensitive and non-radioactive way to detect the activation of transcription factors in samples. In this assay, double stranded DNA sequence containing the NF-kappaB p65 consensus binding site is coated on the 96-well plate. Active NF-kappaB p65 in the cell lysate or the nuclear extract binds to the oligonucleotides on the plate. After the addition of RelA/p65 primary antibody that recognizes the NF-kappaB p65-oligonucleotide complex, a HRP-conjugated secondary antibody is added followed by the addition of TMB substrate and a color signal is developed, which is measured at 450 nm.
PlatformPre-coated microplate (12 x 8 well strips)
Storage instructionsStore at -80°C. Please refer to protocols.
Components 100 tests Antibody Diluent Buffer 1 x 20ml Binding Buffer (5X) 1 x 2.2ml Competitor Oligo (20 pmole) 1 x 25µl DTT (100 mM) 1 x 100µl HRP Conjugate Stock 1 x 8µl Non-Competitor Oligo (20 pmole) 1 x 25µl Plate Coated with DNA Probes 1 unit Plate Sealing Film 2 units Positive Control 1 x 50µl Protease Inhibitor Cocktail 1 x 20µl RelA/p65 Primary Antibody 1 x 200µl Stop Solution 1 x 6ml TMB substrate (Avoid light) 1 x 10ml Wash Buffer (10X) 1 x 27ml
FunctionNF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.
Sequence similaritiesContains 1 RHD (Rel-like) domain.
Domainthe 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
modificationsUbiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'.
Cellular localizationNucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction.
- Information by UniProt
- Avian reticuloendotheliosis viral (v rel) oncogene homolog A
- NF kappa B p65delta3
ab289845 has not yet been referenced specifically in any publications.