Anti-NF-kB p65 antibody - ChIP Grade (ab7970)
- Product nameAnti-NF-kB p65 antibody - ChIP GradeSee all NF-kB p65 primary antibodies ...
- DescriptionRabbit polyclonal to NF-kB p65 - ChIP Grade
- SpecificityReacts with NF-kB p65
- Tested applicationsChIP, ICC, ChIP/Chip, EMSA, IP, IHC-P, WB, IHC-Fr, ICC/IF, Flow Cyt more details
- Species reactivityReacts with: Mouse, Rat, Human
- Positive control
- K-562, NIH/3T3, Hela and A431 cell lysates human DLBCL and human inflammatory skin biopsy
- General notes
Members of the rel/NFkappaB family of transcription factors are involved in the regulation of cellular responses, such as growth, development, and the inflammatory response. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Complexes of p50 (NF-kB1) or p52 (NF-kB2) are generated through the processing of p105 and p100 precursors, respectively. These are usually associated with members of the Rel family (p65, c-Rel, Rel B). The homo- and heterodimer formed through combinations of NF-kB/Rel proteins bind distinct kB sites to regulate the transcription of different genes.
- 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: 0.1% Sodium azide
Constituents: 0.2% Gelatin, PBS
- Concentration information loading...
- PurityAffinity Purified
- Clonality Polyclonal
Our Abpromise guarantee covers the use of ab7970 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||ChIP: Use at an assay dependent dilution.|
|ICC||ICC: Use at an assay dependent concentration.|
|ChIP/Chip||ChIP/Chip: Use at an assay dependent concentration.|
|EMSA||EMSA: Use a concentration of 1 µg/ml.|
|IP||IP: Use at an assay dependent dilution.|
|IHC-P||IHC-P: 1/1000 - 1/4000. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.|
|WB||WB: 1/400 - 1/1000. Detects a band of approximately 64 kDa (predicted molecular weight: 69 kDa).Can be blocked with NF-kB p65 peptide (ab8005).|
|IHC-Fr||IHC-Fr: 1/100. PubMed: 17003500|
|ICC/IF||ICC/IF: Use at an assay dependent dilution. PubMed: 16818736|
|Flow Cyt||Flow Cyt: Use at an assay dependent concentration.|
- 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 antibody - ChIP Grade images
ab7970 used undiluted for ChIP analysis of mouse primary T cell nuclear lysate, incubated for 16 hours at 4°C. Cross-linking (X-ChIP) using formaldehyde for 10 minutes.
Detection step: Real-time PCR.Negative control: Rabbit IgG.
Results showed the dramatic enrichment of p65 binding upon P+I stimulation in IL-4 promoter region and also IL-2 promoter region.
ICC/IF image of ab7970 stained HEK293 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 (ab7970, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 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.
ab7970 staining NF-kB p65 in Human AGS cells by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with formaldehyde, permeabilzed with 0.025% Trton X-100 in TBS and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/100) for 1 hour at 25°C. An Alexa Fluor®568-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
All lanes : Anti-NF-kB p65 antibody - ChIP Grade (ab7970)
Lane 1 : A431 whole cell lysate
Lane 2 : Hela cell lysate
Performed under reducing conditions.
Predicted band size : 69 kDa
Observed band size : 64 kDa (why is the actual band size different from the predicted?)
Western blotting starting dilution: 1/400-1/1000.
Anti-NF-kB p65 antibody - ChIP Grade (ab7970) at 1/1000 dilution + Lysate prepared from mouse lung tissue at 20 µg
Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/20000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 69 kDa
Observed band size : 69 kDa
Exposure time : 15 seconds
This image is a courtesy of Anonymous Abreview
Immunoperoxidase staining of formalin-fixed, paraffin-embedded human DLBCL showing nuclear expression of NF-kB p65 in the tumor cells.
ab7970 staining NFkB p65 in Human stomach tissue sections by IHC-Fr (Immunohistochemistry - Froxen sections). Tissue samples were fixed with acetone and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody 1/1000 in blocking buffer for 1 hour at 25°C. An undiluted HRP-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody.
ab7970 staining NF-kB p65 in mouse lung tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent xylene fixation before heat mediated antigen retrieval in citrate and then blocking with 1.5% serum was performed for 30 minutes at 37°C. The primary antibody was used diluted 1/500 and incubated with sample for 16 hours at 4°C. A Biotin conjugated polyclonal to rabbit IgG was used undiluted as secondary antibody. ABC staining system was used.
References for Anti-NF-kB p65 antibody - ChIP Grade (ab7970)
This product has been referenced in:
- Ai J et al. MicroRNA-195 protects against dementia induced by chronic brain hypoperfusion via its anti-amyloidogenic effect in rats. J Neurosci 33:3989-4001 (2013). WB, ChIP ; Rat . Read more (PubMed: 23447608) »
- Jing J et al. Role of macrophage receptor with collagenous structure in innate immune tolerance. J Immunol 190:6360-7 (2013). Read more (PubMed: 23667110) »