Recombinant
RabMAb

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

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 within Human NF-kB p65 aa 250-350 (phospho S276). The exact sequence is proprietary.
    Database link: Q04206

  • 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

    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.

Properties

Applications

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).

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

  • 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


    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).

  • 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.

  • 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.

References

ab183559 has not yet been referenced specifically in any publications.

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