Overview

  • Product nameAnti-NF-kB p65 antibody [E379]
    See all NF-kB p65 primary antibodies
  • Description
    Rabbit monoclonal [E379] to NF-kB p65
  • Specificityab32536 recognises NF-kB p65.
  • Tested applicationsWB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Human

    Does not react with

    Rat
  • Immunogen

    Synthetic peptide corresponding to Human NF-kB p65 (C terminal).

  • Positive control
    • Hela cell lysate Human Breast carcinoma HeLa cells
  • General notes

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    A 40 µl trial size is available to purchase for this antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32536 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/50000 - 1/100000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
IHC-P Use at an assay dependent dilution.
ICC/IF 1/250 - 1/500.
Flow Cyt 1/100.
IP 1/200.

Target

  • 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.
  • Post-translational
    modifications
    Ubiquitinated, 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.
  • Information by UniProt
  • Database links
  • Alternative names
    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
    • MGC131774 antibody
    • NF kappa B p65delta3 antibody
    • NFKB 3 antibody
    • NFKB3 antibody
    • Nuclear Factor NF Kappa B p65 Subunit antibody
    • Nuclear factor NF-kappa-B p65 subunit antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 antibody
    • Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B Cells antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 antibody
    • OTTHUMP00000233473 antibody
    • OTTHUMP00000233474 antibody
    • OTTHUMP00000233475 antibody
    • OTTHUMP00000233476 antibody
    • OTTHUMP00000233900 antibody
    • p65 antibody
    • p65 NF kappaB antibody
    • p65 NFkB antibody
    • relA antibody
    • TF65_HUMAN antibody
    • Transcription factor p65 antibody
    • v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)) antibody
    • V Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A antibody
    • v rel reticuloendotheliosis viral oncogene homolog A (avian) antibody
    • V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65 antibody
    • v-rel reticuloendotheliosis viral oncogene homolog A antibody
    see all

Anti-NF-kB p65 antibody [E379] images


  • 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.

    See Abreview

  • 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.

  • Anti-NF-kB p65 antibody [E379] (ab32536) at 1/100000 dilution + HeLa cell lysate

    Predicted band size : 65 kDa
    Observed band size : 65 kDa
  • Immunohistochemical analysis of paraffin-embedded human Breast carcinoma using anti-NF-kB p65 Rabbit Monoclonal AntibodyImmunohistochemical analysis of paraffin-embedded human Breast carcinoma using anti-NF-kB p65 Rabbit Monoclonal Antibody
  • Immunofluorescent staining of HeLa cells using anti-NF-kB p65 Rabbit Monoclonal Antibody (ab32536)
  • 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:
  • Yan M  et al. IKKa restoration via EZH2 suppression induces nasopharyngeal carcinoma differentiation. Nat Commun 5:3661 (2014). Read more (PubMed: 24739462) »
  • Manganaro L  et al. Tumor Suppressor Cylindromatosis (CYLD) Controls HIV Transcription in an NF-?B-Dependent Manner. J Virol 88:7528-40 (2014). Human . Read more (PubMed: 24760882) »

See all 10 Publications for this product

Product Wall

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - 70% methanol
Sample Human Cell (MCF-7)
Specification MCF-7
Gating Strategy Live cells gated
Preparation Cell harvesting/tissue preparation method: Cell dissociation buffer
Sample buffer: Enzyme free
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Submitted Aug 15 2014

Application Western blot
Loading amount 100000 cells
Gel Running Conditions Reduced Denaturing (10% gel)
Sample Human Cell lysate - whole cell (MCF-7)
Specification MCF-7
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Aug 12 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (8%)
Sample Mouse Cell lysate - whole cell (mouse embryonic fibroblasts (MEFs))
Specification mouse embryonic fibroblasts (MEFs)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Feb 21 2014

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization Yes - 0.5% Triton X100 in PBS
Fixative Paraformaldehyde
Username

Dr. Kirk McManus

Verified customer

Submitted Jan 15 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Rat Tissue lysate - whole (aorta)
Specification aorta
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Abcam user community

Verified customer

Submitted Oct 23 2013

For HeLa cells, fix and permeabilize with acetone at -20°C for 5 minutes. This treatment results in high quality immunofluorescence images. It is not necessary to permeablize with 0.1% Triton-X100. Mainly nuclear staining is observed. NF-kappaB is also...

Read More

Flow cytometry protocols are recommended below. For most of the antibodies we are not able to provide specific epitope information but it seems most targets are intracellular. Hence not on the membrane and cells need fixation prior to staining. ab2...

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