Anti-NFkB p65 (acetyl K310) antibody - ChIP Grade (ab19870)
- Product nameAnti-NFkB p65 (acetyl K310) antibody - ChIP GradeSee all NFkB p65 primary antibodies ...
- DescriptionRabbit polyclonal to NFkB p65 (acetyl K310) - ChIP Grade
- Tested applicationsWB, IP, Dot Blot, ICC, ChIP more details
- Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide conjugated to KLH derived from within residues 300 - 400 of Human NFkB p65, acetylated at K310.
(Peptide available as ab20612.)
- Positive controlThis antibody gave a positive signal in Rat lung tissue lysate.
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
- Concentration information loading...
- PurityImmunogen affinity 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 NFkB p65 peptide - acetyl K310 (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-NFkB p65 (acetyl K310) antibody - ChIP Grade images
Rabbit polyclonal to NFkB 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 NFkB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for3 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 NFkB 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. No signal is observed in the input as only 2% of the nuclear extract was used for the immunoprecipitation, and the protein might not be abundant.
1. pRRL + TNF-alpha 2. Wt HDACi + TNF-alpha
3. K310R HDACi + TNF-alpha 4. pRRL HDACi + TNF-alpha
5. Wt HDACi + TNF-alpha 6. K310R HDACi + TNF-alpha
All lanes : Anti-NFkB 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
Anti-NFkB 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-NFkB p65 (acetyl K310) antibody - ChIP Grade (ab19870)
This product has been referenced in:
- Kim JW et al. New Molecular Bridge between RelA/p65 and NF-?B Target Genes via Histone Acetyltransferase TIP60 Cofactor. J Biol Chem 287:7780-91 (2012). WB, ChIP . Read more (PubMed: 22249179) »
- Breitenstein A et al. Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells. Cardiovasc Res 89:464-72 (2011). WB ; Human . Read more (PubMed: 20978007) »