Key features and details
- Assay type: Semi-quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr 30 min
- Sample type: Nuclear Extracts
Product nameNFkB p65 Transcription Factor Assay Kit
See all NF-kB p65 kits
Sample typeNuclear Extracts
Assay time3h 30m
Species reactivityReacts with: Mouse, Rat, Human
NFkB p65 Transcription Factor Assay Kit ab133112 is a non-radioactive, sensitive ELISA-based method for detecting specific transcription factor DNA binding activity in nuclear extracts.
In the NFkB p65 assay, a double stranded DNA sequence containing the NFkB response element is immobilized onto the bottom of the wells of a 96-well plate. NFkB contained in a nuclear extract, binds to the NFkB response element, and is detected using an anti NFkB p65 antibody. A secondary antibody conjugated to HRP is added to provide a colorimetric readout at 450 nm.
NFkB p65 transcription factor assay protocol summary:
- prepare nuclear extracts from cells
- add samples to wells
- incubate for 1 hr or o/n
- wash with wash buffer
- add NFkB antibody and incubate for 1 hr , then wash
- add HRP-conjugated secondary antibody and incubate for 1 hr, then wash
- add developing solution and incubate for 15-45 min
- add stop solution
- analyze with microplate reader
Storage instructionsPlease refer to protocols.
Components 96 tests 96-Well Plate Cover 1 unit Polysorbate 20 1 vial Transcription Factor Antibody Binding Buffer (10X) 1 x 3ml Transcription Factor Binding Assay Buffer (4X) 1 x 3ml Transcription Factor Developing Solution 1 x 12ml Transcription Factor Goat Anti-Rabbit HRP Conjugate 1 x 100µl Transcription Factor NFkB (Human p65) Positive Control 1 vial Transcription Factor NFkB (p65) Primary Antibody 1 vial Transcription Factor NF-kB 96-Well Strip Plate 1 unit Transcription Factor NFkB Competitor dsDNA 1 vial Transcription Factor Reagent A 1 x 120µl Transcription Factor Stop Solution 1 x 12ml Wash Buffer Concentrate (400X) 1 x 5ml
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
sELISA pair antibody
After the treatment with LPS (10 μg/ml for 6 hrs), cells were lysed with hypotonic HEPES lysis buffer (pH 7.4) and centrifuged at 1000 g for 10 min at 4°C, supernatants were collected and used for the determination of intracellular p65- NF-κB by ELISA. The absorbance was read at 450 nm using spectrophotometer.
Jurkat cells were treated with PMA and ionomycin (+). Nuclear lysates were extracted (ab113474) and 40 uL, corresponding to 4e6 cells, were tested in duplicates (+/- SD).
Titration of positive control with or without inhibitor, background signal subtracted (duplicates; +/- SD).
Assay of cell lysates isolated from stimulated (20 ng/ml TNF alpha for 30 minutes) and nonstimulated HeLa cells demonstrating NFkB (p65) activity.
ab133112 has been referenced in 40 publications.
- Lin N et al. Bone marrow-derived mesenchymal stem cells utilize the notch signaling pathway to induce apoptosis of hepatic stellate cells via NF-?B sensor. Biol Chem 401:505-515 (2020). PubMed: 31527287
- Yang X et al. FURIN Expression in Vascular Endothelial Cells Is Modulated by a Coronary Artery Disease-Associated Genetic Variant and Influences Monocyte Transendothelial Migration. J Am Heart Assoc 9:e014333 (2020). PubMed: 32067586
- Muri J et al. Thioredoxin-1 distinctly promotes NF-?B target DNA binding and NLRP3 inflammasome activation independently of Txnip. Elife 9:N/A (2020). PubMed: 32096759
- Zhang Y et al. Constitutive activation of the canonical NF-?B signaling pathway in EBV-associated gastric carcinoma. Virology 532:1-10 (2019). PubMed: 30974373
- Zhang G et al. The TLR-4/NF-?B signaling pathway activation in cochlear inflammation of rats with noise-induced hearing loss. Hear Res 379:59-68 (2019). PubMed: 31096078