Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab52175)
- 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
- Specificityab52175 detects endogenous levels of NF-kB p65 only when acetylated at lysine 310.
- Tested applicationsIHC-P, WB, ELISA, ChIP, ICC/IF more details
- Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide corresponding to Human NF-kB p65 (internal sequence) (acetyl K310).
- Positive control
- Extracts from HeLa cells treated with TSA (40nM, 24hours),human lung carcinoma tissue
- Storage instructionsStore at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 50% Glycerol, PBS (without Mg2+ and Ca2+), 150mM Sodium chloride, pH 7.4
- Concentration information loading...
- PurityProtein A purified
- Purification notesab52175 was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific acetylated peptide. The antibody against non-acetylated peptide was removed by chromatography using non-acetylated peptide corresponding to the acetylation site.
- Clonality Polyclonal
Our Abpromise guarantee covers the use of ab52175 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||IHC-P: 1/50 - 1/100.|
|WB||WB: 1/500 - 1/1000. Detects a band of approximately 70 kDa (predicted molecular weight: 60 kDa).|
|ChIP||ChIP: Use at an assay dependent concentration.|
|ICC/IF||ICC/IF: Use a concentration of 1 - 5 µg/ml.|
- 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
ab52175 used undiluted in ChIP.
Whole tissue lysate prepared from murine sciatic nerves.10 seconds cross-linking (X-ChIP) with paraformaldehyde.
Detection step: Semiquantitative PCR.
ICC/IF image of ab52175 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52175, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Immunofluorescence analysis of ab52175 stained HeLa cells.The picture on the right is treated with the synthesized peptide.
Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab52175) + Extracts from HeLa cells,trated with TSA 40nM 24h
Predicted band size : 60 kDa
All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab52175) at 1/500 dilution
Lane 1 : HeLa cell extract treated with TSA (40nM, 24hours) with immunising peptide
Lane 2 : HeLa cell extract treated with TSA (40nM, 24hours)
Alkaline Phosphatase-conjugated Monoclonal Mouse Anti-Rabbit.
Predicted band size : 60 kDa
Observed band size : 70 kDa (why is the actual band size different from the predicted?)
developed using the ECL technique
Predicted band size : 60 kDa
Exposure time : 30 seconds
Image courtesy of Tom Hu by Abreview.
ab52175 used at a 1/1000 dilution in Western Blot.Whole tissue lysate prepared from murine sciatic nerves was loaded at 200µg.Performed under reduced, denaturing conditions.Blocking step 5% milk for 1 hour at 20°C.Secondary used was an HRP conjugated goat anti-rabbit IgG polyclonal used at a 1/5000 dilution.
Immunohistochemistry analysis of paraffin-embedded Human lung carcinoma tissue with ab52175.The picture on the right is treated with synthesized peptide.
References for Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab52175)
This product has been referenced in:
- Ziesché E et al. The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-?B. Nucleic Acids Res 41:90-109 (2013). Read more (PubMed: 23087373) »
- Price LC et al. Nuclear Factor ?-B Is Activated in the Pulmonary Vessels of Patients with End-Stage Idiopathic Pulmonary Arterial Hypertension. PLoS One 8:e75415 (2013). IHC-Fr ; Human . Read more (PubMed: 24124488) »