Validated using a knockout cell line
Recombinant
RabMAb

Anti-NF-kB p65 antibody [E379] (ab32536)

Overview

  • Product name
    Anti-NF-kB p65 antibody [E379]
    See all NF-kB p65 primary antibodies
  • Description
    Rabbit monoclonal [E379] to NF-kB p65
  • Host species
    Rabbit
  • Specificity
    ab32536 recognises NF-kB p65. The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse. This antibody is unsuitable for detecting NF-KB p65 in mouse tissue lysates. The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230. Mouse brain and spleen are positive reported by PMID: 21479220 and 20008488
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human NF-kB p65 aa 500 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: Q04206

  • Positive control
    • WB: HeLa cell lysate. IHC-P: Human breast carcinoma tissue. ICC/IF: HeLa cells.
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal 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).

This antibody might not detect target band in mouse tissues.

IHC-P 1/2000.

The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse.

ICC/IF 1/100.

For unpurified use at 1/250 -1/500.

Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

For unpurified use at 1/100.

IP 1/30.

For unpurified use at 1/200. 

Target

  • Function
    NF-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 similarities
    Contains 1 RHD (Rel-like) domain.
  • Domain
    the 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 localization
    Nucleus. 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
    • 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 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
    see all

Images

  • ab32536 (purified) at 1:30 dilution (2μg) immunoprecipitating NF-kB p65 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

    Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
    Lane 2 (+): ab32536 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32536 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

    For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-Kb p65 with purified ab32536 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor ® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab32536 at 1:100 dilution. Cells were fixed in 100% Methanol. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon carcinoma tissue sections labeling NF-kB p65 with Purified ab32536 at 1:2000 dilution (0.2 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

  • Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 65 kDa



    Blocking and diluting buffer: 5% NFDM/TBST.

  • Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution (purified) + MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 65 kDa



    Blocking and diluting buffer: 5% NFDM/TBST.

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: NF-kB p65 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: A431 cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab32536 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.


    Unpurified ab32536 was shown to specifically react with NF-kB p65 in wild-type HAP1 cells. No band was observed when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 (NF-kB p65) and ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  • Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with unpurified 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.

  • Immunofluorescent staining of HeLa cells using anti-NF-kB p65 Rabbit Monoclonal Antibody ( unpurified ab32536)

  • Overlay histogram showing MCF-7 cells stained with unpurified 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.

  • Anti-NF-kB p65 [E379] antibody (unpurified 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 unpurified 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

  • Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536. Briefly, the tested cells were seeded on coverslips treated with HCl and ethanol, and autoclaved prior to use. Immunostaining of the p65 subunit of NF-κB was done by permeabilizing the cells with Triton X-10, then by treating the cells with anti-NF-κB p65 rabbit monoclonal primary antibody [E379] (ab32536), followed by Alexa Fluor® 488 Donkey anti-rabbit IgG secondary antibody. Nuclei of cells were stained with DAPI. Images were acquired using fluorescence microscope. 

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

    Predicted band size: 65 kDa
    Observed band size: 65 kDa

  • Immunohistochemical analysis of paraffin-embedded human Breast carcinoma using unpurified anti-NF-kB p65 Rabbit Monoclonal Antibody

  • All lanes : Anti-NF-kB p65 antibody [E379] (ab32536) at 1000 µg

    Lane 1 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
    Lane 2 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharides for 6 hours whole cell lysates
    Lane 3 : Mouse brain lysates
    Lane 4 : Mouse spleen lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 65 kDa


    Exposure time: 3 seconds


    This antibody is unsuitable for detecting mouse tissue lysates.

    The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230.

    Mouse brain and spleen are positive reported by PMID: 21479220 and 20008488

References

This product has been referenced in:
  • Wen H  et al. Recurrent ECSIT mutation encoding V140A triggers hyperinflammation and promotes hemophagocytic syndrome in extranodal NK/T cell lymphoma. Nat Med 24:154-164 (2018). WB . Read more (PubMed: 29291352) »
  • Saito K  et al. Xanthohumol inhibits angiogenesis by suppressing nuclear factor-?B activation in pancreatic cancer. Cancer Sci 109:132-140 (2018). Read more (PubMed: 29121426) »

See all 46 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (mesenchymal stem cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
40 µg
Specification
mesenchymal stem cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Kate Hawkins

Verified customer

Submitted Feb 22 2017

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Colon)
Gel Running Conditions
Reduced Denaturing (12.5%)
Loading amount
1e+006 cells
Specification
Colon
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

Submitted Dec 23 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Reduced Denaturing (12.5%)
Loading amount
1e+006 cells
Specification
HeLa
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

Submitted Dec 23 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (human head and neck cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Treatment
10 nM PMA
Specification
human head and neck cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Aug 31 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (Human THP-1 macrophages)
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Loading amount
70 µg
Treatment
100 µg/ml oxidized LDL (oxLDL) +/- 10 µg/ml apoA-I, 24h
Specification
Human THP-1 macrophages
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Anca Sima

Verified customer

Submitted Jun 04 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Sample
Human Cell (MCF-7)
Specification
MCF-7
Permeabilization
Yes - Triton X-100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 23 2014

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
Username

Abcam user community

Verified customer

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
Username

Abcam user community

Verified customer

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
Username

Abcam user community

Verified customer

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

1-10 of 13 Abreviews or Q&A

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