Product namePhospho-NF-kappaB p65 (S468 + S536) + Total In-Cell ELISA Kit
See all NF-kB p65 kits
Sample typeAdherent cells, Suspension cells
Assay typeCell-based (quantitative)
Assay durationMultiple steps standard assay
Species reactivityReacts with: Human
Phospho-NF-kappaB p65 (S468 + S536) + Total In-Cell ELISA Kit (ab207481) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) NF-kappaB p65 and/or total NF-kappaB p65. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated NF-kappaB p65 or total NF-kappaB p65. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.
The Phospho-NF-kappaB p65 (S468 + S536) + Total In-Cell ELISA Kit contains three 96-well plates and three primary antibodies. The phospho-NF-kappaB p65 antibodies are specific for phosphorylated NF-kappaB p65 and do not cross-react with other family members. The phospho-NF-kappaB p65 (S468) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 468 of human NF-kappaB p65 and does not cross-react with other sites. The phospho-NF-kappaB p65 (S536) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 536 of human NF-kappaB p65 and does not cross-react with other sites. The total- NF-kappaB p65 antibody recognizes NF-kappaB p65 proteins regardless of the phosphorylation state. The kit can be used to study phosphorylated NF-kappaB p65 relative to cell number or to determine NF-kappaB p65 phosphorylation relative to the total NF-kappaB p65 protein found in the cells. Once the phospho-NF-kappaB p65 and total-NF-kappaB p65 signals have been normalized for cell number, a comparison of the ratio of phosphorylated NF-kappaB p65 to total NF-kappaB p65 for each of the cell growth conditions can be made. The provided total-NF-kappaB p65 antibody can be used as a positive control to demonstrate that the cells contain NF-kappaB p65, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, many plates can be prepared simultaneously and then the assay performed when desired.
The transcription factor NF-kappaB (nuclear factor kappaB, NFkB) is a key component for the inducible expression of a wide variety of cellular and viral genes. NF-kappaB is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses. The DNA-binding protein complex recognizes a discrete nucleotide sequence (5´-GGGACTTTCC-3´) in the upstream region of a variety of cellular and viral response genes. NF-kappaB is composed of homo- and heterodimeric complexes of members of the Rel (NF-kappaB) family. There are five subunits of the NF-kappaB 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. This domain is also a target of the IKB inhibitors, which include IKB alpha, IKB beta, IKB gamma, Bcl-3, p105 and p100. The p50/p65 (NF-kappaB1/RelA) heterodimers and the p50 homodimers are the most common dimers found in the NF-kappaB signaling pathway. In the majority of cells, NF-kappaB 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. During the phosphorylation and degradation of IKBs, NF-kappaB p65 is phosphorylated on multiple residues, each triggered by different stimuli but essential for maintaining NF-kappaB transcriptional activation. Phosphorylation at Ser536 occurs in the C-terminal transactivation domain and is critical for p65 transcriptional activity. Phosphorylation at this site is triggered by several stimuli including phorbol ester, IL-1 alpha and TNF-alpha as mediated by IKB kinase and p38 MAPK. Phosphorylation at Ser276 by PKA and/or MSK1 enables increased interaction with the transcriptional coactivator p300/CBP to further enhance the transcriptional activity of NF-kappaB. In contrast, PMA-induced NF-kappaB transcriptional activity is dependent on the region between amino acids 442 and 470, suggesting a role for one or more of the potential phosphorylation sites (Ser457, Thr458, Thr464 or Ser468).
Tested applicationsSuitable for: In-Cell ELISAmore details
Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 1% SDS Solution 2 x 22ml 10% Triton X-100 1 x 10ml 10X PBS 2 x 120ml 1X Antibody Blocking Buffer 2 x 22ml 1X Antibody Dilution Buffer 2 x 30ml 96-well tissue culture plate 3 units Anti-rabbit HRP-conjugated IgG 2 x 11µl Crystal Violet Solution 2 x 22ml Developing Solution 3 x 11ml Phospho-NFkB p65 (Ser468) antibody 1 x 9µl Phospho-NFkB p65 (Ser536) antibody 1 x 9µl Plate sealer 3 units Stop Solution 3 x 11ml Total-NFkB antibody 1 x 9µl
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
Our Abpromise guarantee covers the use of ab207481 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||Use at an assay dependent concentration.|
HeLa cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20 ng/ml TNF-α and 50 nM Calyculin A for 15 minutes and fixed. Total and phospho NFκB p65 were each assayed in triplicate using the phospho and total NFκB p65 antibodies included in the kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).
ab207481 has not yet been referenced specifically in any publications.