Anti-NF-kB p65 antibody (ab16502)
- Product nameAnti-NF-kB p65 antibodySee all NF-kB p65 primary antibodies ...
- DescriptionRabbit polyclonal to NF-kB p65
- Tested applicationsIHC-FoFr, ICC/IF, IHC-Fr, ICC, IHC-P, WB, IP more details
- Species reactivityReacts with: Mouse, Rat, Chicken, Human, Indian Muntjac, Other
Synthetic peptide corresponding to Human NF-kB p65 aa 500 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
(Peptide available as
- Positive control
- This antibody gave a positive signal in the following lysates: Mouse Spleen Tissue, HeLa Whole Cell, A431 Whole Cell
- Storage instructionsStore at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
- Concentration information loading...
- PurityProtein A purified
- Clonality Polyclonal
Our Abpromise guarantee covers the use of ab16502 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||IHC-FoFr: Use at an assay dependent concentration.|
|ICC/IF||ICC/IF: Use a concentration of 1 µg/ml.|
|IHC-Fr||IHC-Fr: Use at an assay dependent concentration.|
|ICC||ICC: Use at an assay dependent concentration.|
|IHC-P||IHC-P: Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||WB: Use a concentration of 0.5 µg/ml. Detects a band of approximately 64 kDa (predicted molecular weight: 60 kDa).Can be blocked with Human NF-kB p65 peptide (ab16636).|
|IP||IP: Use at an assay dependent concentration.|
- 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.
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Anti-NF-kB p65 antibody images
IHC image of NF-kB p65 staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16502, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
All lanes : Anti-NF-kB p65 antibody (ab16502) at 1 µg/ml
Lane 1 : Spleen (Mouse) Tissue Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 60 kDa
Observed band size : 64 kDa (why is the actual band size different from the predicted?)
Exposure time : 8 minutes
NF-kB p65 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to NFkB p65 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16502.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 68kDa: NFkB p65
ab16502 stining the nuclei of the cardiac cells in rat tissue. The tissues were fixed (animals perfused fixed) with 4% PFA and later postfixed overnight in the same fixative. They were cryoprotected in 30% sucrose and cut using a cryostat.
ICC/IF image of ab16502 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16502, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1:200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
ab16502 at a 1/500 dilution staining Asynchronous and paraformaldehyde-fixed (4%) HeLa cells by immunocytochemistry. The antibody was incubated with the cells 30 minutes and then detected using a Cy3 conjugated Goat Anti-Mouse IgG (H+L) antibody.
This image is courtesy of an Abreview by Kirk McManus submitted on 27 February 2006.
ICC/IF image of ab16502 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16502, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 phalloidin was used to label F-actin (red).
References for Anti-NF-kB p65 antibody (ab16502)
This product has been referenced in:
- Ganapathy S et al. Low-dose arsenic induces chemotherapy protection via p53/NF-?B-mediated metabolic regulation. Oncogene N/A:N/A (2013). Read more (PubMed: 23524579) »
- Abraham R et al. Induction of cytopathogenicity in human glioblastoma cells by chikungunya virus. PLoS One 8:e75854 (2013). WB . Read more (PubMed: 24086645) »