Product nameAnti-NF-kB p65 (phospho S276) antibody [EPR17622]
See all NF-kB p65 primary antibodies
DescriptionRabbit monoclonal [EPR17622] to NF-kB p65 (phospho S276)
Tested applicationsSuitable for: ICC/IF, IP, WBmore details
Species reactivityReacts with: Human
Synthetic peptide within Human NF-kB p65 aa 250-350 (phospho S276). The exact sequence is proprietary.
Database link: Q04206
- WB: Lysates of HeLa cells treated with Calyculin A (100ng/ml, 30min), then TNF-a (20ng/ml, 5min). ICC/IF: HeLa cells treated with Calyculin A (100ng/ml, 10min), then TNF-a (20ng/ml, 5min). IP: HeLa whole cell lysate treated with Calyculin A (100ng/ml, 10min), then TNF-a (20ng/ml, 5min).
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage bufferPreservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
PurityProtein A purified
Our Abpromise guarantee covers the use of ab183559 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/1000. Detects a band of approximately 80 kDa (predicted molecular weight: 60 kDa).|
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
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All lanes : Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559) at 1/1000 dilution
Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes
Lane 3 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes, then treated with Alkaline Phosphatase for 1 hour
Lysates/proteins at 10 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 60 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?
Exposure time: 3 minutes
Blocking/Dilution buffer: 5% NFDM/TBST.
Phosphorylation of NF-kB p65 at S276 can be induced by TNF-a treatment according to the literature (PMID: 21795584).
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling NF-kB p65 (phospho S276) with ab183559 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).
The expression increased on HeLa cells after treatment with Calyculin A (ab141784 100ng/ml, 10min) then TNF-a (20ng/ml, 5min).
The nuclear counter stain is DAPI (blue).
Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.
NF-kB p65 (phospho S276) was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates, treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min, with ab183559 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183559 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min, 10 μg (Input).
Lane 2: ab183559 IP in HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab183559 in HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.
ab183559 has not yet been referenced specifically in any publications.