Recombinant
RabMAb

Recombinant Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533)

Overview

  • Product name

    Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade
    See all NF-kB p65 primary antibodies
  • Description

    Rabbit monoclonal [EPR21781] to NF-kB p65 (acetyl K310) - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, Dot blot, ChIPmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human NF-kB p65 aa 300-400 (acetyl K310). The exact sequence is proprietary.
    Database link: Q04206

  • Positive control

    • WB: HEK-293 transfected with NF-kB p65 and p300 (aa1287-1663) expression vectors containing a myc-His-tag®, then treated with 20 ng/ml TNF-alpha (ab9642) for 60 minutes whole cell lysate; Human lung tissue lysate. ChIP: Chromatin prepared from HeLa cells.
  • 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 ab218533 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 1/1000. Detects a band of approximately 70 kDa (predicted molecular weight: 60 kDa).
Dot blot 1/1000.
ChIP Use 2 µg for 25 µg of chromatin.

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

  • Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533) at 1/1000 dilution + Human lung tissue lysate at 20 µg

    Secondary
    VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

    Predicted band size: 60 kDa
    Observed band size: 65 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

    We used a high sensitivity ECL substrate to develop this blot.

  • Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with and without 20 ng/ml TNF-α (ab9642) for 60 minutes according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 5 μg of ab218533 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).

    The ChIP data are consistent with the literature (PMID: 16135789).

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533) at 1/2000 dilution

    Lane 1 : HEK-293 transfected with NF-kB p65 expression vector containing a myc-His-tag®, whole cell lysate
    Lane 2 : HEK-293 transfected with NF-kB p65 and p300 (aa1287-1663) expression vectors containing a myc-His-tag®, then treated with 20 ng/ml TNF-alpha (ab9642) for 60 minutes, whole cell lysate

    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: 70 kDa why is the actual band size different from the predicted?


    Exposure time: 37 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    NF-κB p65 (acetyl K310) expression is induced by TNF-α and p300 acetyltransferases (PMID: 20160011, PMID: 12456660, PMID: 16135789).

  • Dot blot analysis of NF-kB p65 (acetyl K310) labeled with ab218533 at 1/1000 dilution.

    Lane 1: NF-κB p65 (acetyl K310) peptide.
    Lane 2: NF-κB p65 non-acetyl peptide.

    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: 2 minutes.

References

ab218533 has not yet been referenced specifically in any publications.

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