RecombinantRabMAb

Recombinant Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)

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Western blot - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
  • Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
  • Immunoprecipitation - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
  • Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR17622] to NF-kB p65 (phospho S276)
  • Suitable for: ICC/IF, IP, WB
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-NF-kB p65 (phospho S276) antibody [EPR17622]
    See all NF-kB p65 primary antibodies

  • Description

    Rabbit monoclonal [EPR17622] to NF-kB p65 (phospho S276)

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, IP, WBmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Lysates of HeLa cells treated with Calyculin A (100ng/ml, 30min), then TNF-a (20ng/ml, 5min). ICC/IF: HeLa cells treated with Calyculin A (100ng/ml, 10min), then TNF-a (20ng/ml, 5min). IP: HeLa whole cell lysate treated with Calyculin A (100ng/ml, 10min), then TNF-a (20ng/ml, 5min).

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab183559 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
ICC/IF
1/100.
IP
1/40.
WB
1/1000. Detects a band of approximately 80 kDa (predicted molecular weight: 60 kDa).
Notes
ICC/IF
1/100.
IP
1/40.
WB
1/1000. Detects a band of approximately 80 kDa (predicted molecular weight: 60 kDa).

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

    • Western blot - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
      Western blot - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
      All lanes : Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559) at 1/1000 dilution

      Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
      Lane 2 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes
      Lane 3 : HeLa whole cell lysate treated with 100ng/ml Calyculin A ab141784 for 30 minutes, then treated with 20ng/ml TNF-a for 5 minutes, then treated with Alkaline Phosphatase for 1 hour

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 60 kDa
      Observed band size: 80 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Negative control: Anti-NF-kB p65 antibody, ab32536

      Blocking/Dilution buffer: 5% NFDM/TBST.

      Phosphorylation of NF-kB p65 at S276 can be induced by TNF-a treatment according to the literature (PMID: 21795584).

       

    • Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
      Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling NF-kB p65 (phospho S276) with ab183559 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

      The expression increased on HeLa cells after treatment with Calyculin A (ab141784 100ng/ml, 10min) then TNF-a (20ng/ml, 5min).

      The nuclear counter stain is DAPI (blue).

      Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

    • Immunoprecipitation - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
      Immunoprecipitation - Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)

      NF-kB p65 (phospho S276) was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates, treated with 100ng/ml Calyculin A (ab141784) for 10min, then 20ng/ml TNA-a for 5min, with ab183559 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183559 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

      Lane 1: HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min, 10 μg (Input).

      Lane 2: ab183559 IP in HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab183559 in HeLa whole cell lysate treated with 100ng/ml CA for 10min, then 20ng/ml TNA-a for 5min.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 10 seconds.

    • Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)
      Anti-NF-kB p65 (phospho S276) antibody [EPR17622] (ab183559)

    References (4)

    Publishing research using ab183559? Please let us know so that we can cite the reference in this datasheet.

    ab183559 has been referenced in 4 publications.

    • Li S  et al. Tumor-educated B cells promote renal cancer metastasis via inducing the IL-1ß/HIF-2a/Notch1 signals. Cell Death Dis 11:163 (2020). PubMed: 32123166
    • Okasha SM  et al. Sirtuin 1 Activation Suppresses the Growth of T-lymphoblastic Leukemia Cells by Inhibiting NOTCH and NF-?B Pathways. Anticancer Res 40:3155-3161 (2020). PubMed: 32487610
    • Ouyang XL  et al. NF-?B inhibitory and cytotoxic activities of hexacyclic triterpene acid constituents from Glechoma longituba. Phytomedicine 63:153037 (2019). PubMed: 31357075
    • Li Q  et al. Ethanol Exposure Induces Microglia Activation and Neuroinflammation through TLR4 Activation and SENP6 Modulation in the Adolescent Rat Hippocampus. Neural Plast 2019:1648736 (2019). PubMed: 31781182

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