The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/50000 - 1/100000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
Use at an assay dependent concentration.
1/250 - 1/500.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
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.
Domainthe 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
Post-translational 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 [E379] images
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Predicted band size : 65 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: NF-kB 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 - ab32536 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa. ab32536 was shown to specifically react with NF-kB p65 when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 and ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
Western blot - Anti-NF-kB p65 [E379] antibody (ab32536)Image courtesy of an anonymous Abreview
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 65 kDa Observed band size : 65 kDa Additional bands at : 50 kDa (possible cross reactivity),~90 kDa (possible cross reactivity).
Exposure time : 30 seconds
Image courtesy of an anonymous Abreview
Anti-NF-kB p65 [E379] antibody (ab32536) reactivity with reduced wild type (WT) and p65 knockout (KO) mouse embryonic fibroblast (MEF) lysate. After SDS-PAGE, membranes were blocked in 5% milk in TBS + 0.1% Tween for 1h at 25°C before incubation with ab32536 (1:1,000 dilution in 5% milk TBS + 0.1% Tween) for 16h at 4ºC. Blots was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.
Western blot - Anti-NF-kB p65 [E379] antibody (ab32536)
Anti-NF-kB p65 antibody [E379] (ab32536) at 1/100000 dilution + HeLa cell lysate
Predicted band size : 65 kDa Observed band size : 65 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 [E379] antibody (ab32536)Image from Bel S et al., J Biol Chem. 2012 Jul 20;287(30):25631-9. Epub 2012 May 2. Fig 8.; doi: 10.1074/jbc.M112.364786; July 20, 2012, The Journal of Biological Chemistry, 287, 25631-25639.
Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with ab32536.
Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit.
Overlay histogram showing MCF-7 cells stained with ab32536 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32536, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
References for Anti-NF-kB p65 antibody [E379] (ab32536)
This product has been referenced in:
Lin JJ et al. Toll-like receptor 4 signaling in neurons of trigeminal ganglion contributes to nociception induced by acute pulpitis in rats. Sci Rep5:12549 (2015).
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