Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)

Overview

  • Product name

    Anti-NF-kB p65 antibody [E379] - BSA and Azide free
    See all NF-kB p65 primary antibodies
  • Description

    Rabbit monoclonal [E379] to NF-kB p65 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IP, WB, ICC/IF, IHC-Pmore details
    Unsuitable for: Flow Cyt
  • 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

    • HeLa whole cell lysate (ab150035) Human Breast carcinoma HeLa cells
  • General notes

    ab207297 is the carrier-free version of ab32536 This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Ab207297 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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.

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab207297 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
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
ICC/IF Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
  • Application notes
    Is unsuitable for Flow Cyt.
  • 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
      • nfkappabp65 antibody
      • NFkB p65 antibody
      • NFKB3 antibody
      • Nuclear factor kappaB 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 NFKB3 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

    • This WB data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).

      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.


      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.

    • 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 Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
      Blocking and diluting buffer: 5% NFDM/TBST.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

    • 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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

    • 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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

    • 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. 

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

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

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

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

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

    • This IHC data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).

      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.

    References

    This product has been referenced in:

    • Kim YH  et al. Expression of Nod-like Receptors and Clinical Correlations in Patients with Dry Eye Disease. Am J Ophthalmol N/A:N/A (2019). Read more (PubMed: 30653959) »
    • Cai J  et al. Recombinant fimbriae protein of Porphyromonas gingivalis induces an inflammatory response via the TLR4/NF-?B signaling pathway in human peripheral blood mononuclear cells. Int J Mol Med 43:1430-1440 (2019). Read more (PubMed: 30664173) »
    See all 25 Publications for this product

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