Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP2161Y] to NF-kB p65
- Suitable for: WB, Flow Cyt, ICC/IF
- Knockout validated
- Reacts with: Human
Product nameAnti-NF-kB p65 antibody [EP2161Y]
See all NF-kB p65 primary antibodies
DescriptionRabbit monoclonal [EP2161Y] to NF-kB p65
Specificityab76311 detects both phosphorylated and non-phosphorylated versions of human NF-kB p65.
Tested applicationsSuitable for: WB, Flow Cyt, ICC/IFmore details
Unsuitable for: IHC-P or IP
Species reactivityReacts with: Human
Synthetic peptide within Human NF-kB p65. The exact sequence is proprietary.
- HeLa, Daudi and Jurkat cell lysates.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage bufferpH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.5% BSA
Concentration information loading...
PurityProtein A purified
Our Abpromise guarantee covers the use of ab76311 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 - 1/5000. Detects a band of approximately 70 kDa (predicted molecular weight: 65 kDa).|
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
|ICC/IF||1/100 - 1/500.|
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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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NF-κB p65 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab76311 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.
ab76311 was shown to specifically react with NF-κB p65 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when NF-κB p65 knockout samples were used. Wild-type and NF-κB p65 knockout samples were subjected to SDS-PAGE. ab76311 (NF-κB p65) and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
Immunocytochemistry/Immunofluorescence analysis HT-29 (human colorectal adenocarcinoma) labelling NF-kB p65 with purified ab76311 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).
Control: PBS only
All lanes : Anti-NF-kB p65 antibody [EP2161Y] (ab76311) at 1/20000 dilution
Lane 1 : HeLa cell lysate
Lane 2 : Daudi cell lysate
Lane 3 : Jurkat cell lysate
Lysates/proteins at 10 µg per lane.
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution
Predicted band size: 65 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?
ab76311 has been referenced in 11 publications.
- Li X et al. The Specific Inhibition of SOD1 Selectively Promotes Apoptosis of Cancer Cells via Regulation of the ROS Signaling Network. Oxid Med Cell Longev 2019:9706792 (2019). PubMed: 30911355
- Hsueh YS et al. Nuclear KIT induces a NFKBIB-RELA-KIT autoregulatory loop in imatinib-resistant gastrointestinal stromal tumors. Oncogene 38:6550-6565 (2019). PubMed: 31363162
- Ma M et al. Celecoxib enhances sensitivity to chemotherapy drugs of T-cell lymphoma. Oncol Lett 15:4649-4656 (2018). PubMed: 29541237
- Sikorski K et al. A high-throughput pipeline for validation of antibodies. Nat Methods 15:909-912 (2018). PubMed: 30377371
- Jatczak-Pawlik I et al. Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-?B Pathway in a Myeloid Cell Line. Pharm Res 34:136-147 (2017). PubMed: 27766462