Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302)

Overview

  • Product nameAnti-NF-kB p65 (phospho S536) antibody [EP2294Y]
    See all NF-kB p65 primary antibodies
  • Description
    Rabbit monoclonal [EP2294Y] to NF-kB p65 (phospho S536)
  • Tested applicationsSuitable for: Dot blot, WB, IPmore details
    Unsuitable for: Flow Cyt,ICC/IF or IHC-P
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human NF-kB p65 (phospho S536). The exact sequence is proprietary.
    (Peptide available as ab202905)

  • Positive control
    • WB: HeLa and Daudi cell lysate treated with Calyculin A + TNF-alpha. IP: Daudi cell lysate treated with Calyculin A + TNF-alpha.
  • General notes

    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.

    A trial size is available to purchase for this antibody.

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

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

Properties

Applications

Our Abpromise guarantee covers the use of ab76302 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
Dot blot 1/1000.
WB 1/10000 - 1/20000. Predicted molecular weight: 65 kDa.
IP 1/20 - 1/30.
  • Application notesIs unsuitable for Flow Cyt,ICC/IF or IHC-P.
  • Target

    • FunctionNF-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 similaritiesContains 1 RHD (Rel-like) domain.
    • Domainthe 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 localizationNucleus. 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

    Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] images

    • All lanes : Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302) at 1/10000 dilution (purified)

      Lane 1 : HeLa whole cell lysate - untreated at 25 µg
      Lane 2 : HeLa whole cell lysate - treated with calyculin A and tumor necrosis factor-alpha at 10 µg

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

      Predicted band size : 65 kDa
      Observed band size : 65 kDa
      Blocking and dilution buffer: 5% NFDM/TBST.
    • ab76302 (purified) at 1/30 immunoprecipitating NF-kB p65 (phospho S536) in Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.

      Lane 1 (input): Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha (10µg)

      Lane 2 (+): ab76302 + Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.

      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76302 in Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.

      For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Dot blot analysis of INF- kB p65 (phospho S536) phospho peptide (Lane 1) and NF- kB p65 non-phospho peptide (Lane 2) labeling NF-kB p65 (phospho S536) with ab76302 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/100 000 was used as the secondary antibody.

      Blocking and diluting buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • All lanes : Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302) at 1/20000 dilution (unpurified)

      Lane 1 : Untreated Daudi cell lysate
      Lane 2 : Daudi cell lysate treated with Calyculin A + TNF-alpha

      Lysates/proteins at 10 µg per lane.

      Secondary
      HRP labelled goat anti-rabbit at 1/1000 dilution

      Predicted band size : 65 kDa

    References for Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302)

    This product has been referenced in:
    • Liu H  et al. Atractylenolide I modulates ovarian cancer cell-mediated immunosuppression by blocking MD-2/TLR4 complex-mediated MyD88/NF-?B signaling in vitro. J Transl Med 14:104 (2016). Human . Read more (PubMed: 27118139) »
    • Lin IL  et al. The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NF?B. Cancer Cell Int 14:1 (2014). WB ; Human . Read more (PubMed: 24393431) »

    See all 4 Publications for this product

    Product Wall

    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 Jan 12 2016

    Application Western blot
    Sample Mouse Cell lysate - whole cell (NIH3T3)
    Gel Running Conditions Non-reduced Denaturing (10% seperating gel)
    Loading amount 20 µg
    Treatment 0,15,30,60,120,180 min. LPS
    Specification NIH3T3
    Username

    Miss. WEI-HSIN PENG

    Verified customer

    Submitted Dec 07 2015

    Application Western blot
    Loading amount 10 µg
    Gel Running Conditions Non-reduced Denaturing (10)
    Sample Human Cell lysate - whole cell (PBMCs)
    Specification PBMCs
    Treatment CD40L 500ug/ml for 48hours
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Username

    Abcam user community

    Verified customer

    Submitted Jan 12 2015

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"