Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870)
- Product nameAnti-NF-kB p65 (acetyl K310) antibody - ChIP GradeSee all NF-kB p65 primary antibodies ...
- DescriptionRabbit polyclonal to NF-kB p65 (acetyl K310) - ChIP Grade
- Tested applicationsWB, IP, Dot Blot, ICC, ChIP more details
- Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide corresponding to Human NF-kB p65 aa 300-400 (internal sequence) conjugated to Keyhole Limpet Haemocyanin (KLH).
(Peptide available as
- Positive control
- This antibody gave a positive signal in Rat lung tissue lysate.
- 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 ab19870 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||WB: Use a concentration of 2.5 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).Can be blocked with Human NF-kB p65 (acetyl K310) peptide (ab20612). Collaborator data suggests that immunoprecipitation of this antibody prior to Western blotting is required to obtain the best results (see images)|
|IP||IP: Use a concentration of 2.5 µg/ml.|
|Dot Blot||Dot: Use at an assay dependent dilution.|
|ICC||ICC: Use at an assay dependent dilution. In ICC/IF ab19870 recognizes various acetylated nuclear protein(s), as the signal is also observed in control cells; the signal in ICC is HDACi-dependent.|
|ChIP||ChIP: Use at an assay dependent concentration. PubMed: 22249179|
- 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 (acetyl K310) antibody - ChIP Grade images
Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3h at room temperature. Exposure time: 75 min normal ECL. This Dot blot demonstrates that ab19870 recognized upto 10ng of purified peptide on a PVDF membrane.
Western Blot with ab19870 after p65 Immunoprecipitation: rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3 hours at room temperature. Exposure time: 1 min normal ECL.. Tested samples: nuclear extracts (180 µg) of immortalized p65-/- mouse cells, complemented with the empty vector (pRRL), wild-type p65 (Wt) and non-acetylatable K310 (K310R). The samples tested were treated with deacetylase inhibitors HDACi (TSA + Nicotinamide) and TNF-alpha. The samples were immunoprecipitated with 2µg of alpha-p65 and subsequently analysed by Western blot with Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870). Predicted band size = 65kDa, Observed band size = 75kDa. The p65 band runs higher in this SDS-PAGE blot as it contains a myc-tag.All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 2.5 µg/ml
Lane 1 : pRRL untreated
Lane 2 : pRRL HDACi
Lane 3 : pRRL HDACi + TNF
Lane 4 : Wt untreated
Lane 5 : Wt HDACi
Lane 6 : Wt HDACi + phorbol myristate acetate
Lane 7 : K310R untreated
Lane 8 : K310R HDACi
Lane 9 : K310R HDACi + phorbol myristate acetate
Lysates/proteins at 75 µg per lane.
developed using the ECL technique
Predicted band size : 65 kDa
Observed band size : 75 kDa (why is the actual band size different from the predicted?)
Exposure time : 1 hour
Christine Buerki and Karin Rothgiesser
ab19870 recognizes Rabbit polyclonal to NF-kB p65 (acetyl K310) specifically at ~75kDa (indicated by the arrow) is this SDS-PAGE blot. The p65 band runs higher than 65kDa in this SDS-PAGE blot as it contains a myc-tag. We are sure that the band at ~75kDa is p65 since p65 specific antibodies detect the same band in IP and WB and there is no signal in the p65 knock-out cell line with ab19870. A number of additonal bands are recognized by ab19870 when tested with endogenous p65 from whole cell extracts, we do not know the identity of these bands.
Tested samples: nuclear extracts (75µg) of immortalized p65-/- mouse cells, complemented with the empty veAnti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1 µg/ml + Lung (Rat) Tissue Lysate at 10 µg
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size : 65 kDa
Observed band size : 72 kDa (why is the actual band size different from the predicted?)
Additional bands at : 15 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 4 minutes
References for Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870)
This product has been referenced in:
- Scuto A et al. SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-?B/STAT3 complex to its promoter in malignant lymphoid cells. Cell Death Dis 4:e635 (2013). ChIP ; Human . Read more (PubMed: 23681230) »
- Kania G et al. Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis. Circulation 127:2285-94 (2013). Read more (PubMed: 23671208) »