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PhosphoTracer NFkappaB p65 pSer536 + Total ELISA Kit (ab119664)

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Overview

  • Product namePhosphoTracer NFkappaB p65 pSer536 + Total ELISA Kit
  • Detection methodFluorescent
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts
  • Assay typeSandwich
  • Sensitivity
    10 µg protein/ml
  • Range
    10 µg protein/ml - 1000 µg protein/ml
  • Assay time
    1h 15m
  • Assay durationOne step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    PhosphoTracer assays use a traditional immuno-sandwich format, but with a major difference both the analyte and the assay reagents are added to the PhosphoTracer assay microplate at the same time. After a short incubation period, unbound assay reagents and analytes are washed away, and immuno-complexes containing both antibodies are detected. The process can take as little as 60 minutes to complete.

    PhosphoTracer kits also allow a higher degree of assay flexibility. In contrast to other ELISA formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate. Simply mix the lysate with your target reagents of choice, using the microplate configuration of your choice.

    A whole new way of performing cellular assays, PhosphoTracer takes the hard work out of running a standard ELISA, while still giving the high quality results expected from a sandwich immunoassay. Fully self-contained kits are supplied in convenient 96-well packs. Simple to use and highly sensitive PhosphoTracer kits are designed to get results, fast.

     

    Abcam's PhosphoTracer NFKappaB p65 assay kits detect endogenous levels of NFKappaB p65 (GenBank Accession NP_068810) in cellular lysates. The phospho-NFKappaB p65 assay detects NFKappaB p65 only when phosphorylated at Ser536. The total NFKappaB p65 assay detects NFKappaB p65 irrespective of phosphorylation.



    The substrate used with the HRP conjugated detection antibody is a combination of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (wavelength exc/em = 530-540nm / 590-600nm), a highly sensitive and stable substrate for HRP) and ADHP Dilution Buffer (a stabilized H2O2 solution). Learn more about the fluorogenic substrate, ADHP.

  • Tested applicationsSandwich ELISA more details
  • PlatformMicroplate

Properties

  • 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.

    • Target information above from: UniProt accession Q04206 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
    • Alternative names
        NFKB3Nuclear factor NF-kappa-B p65 subunitNuclear factor of kappa light polypeptide gene enhancer in B-cells 3
        p65relATF65_HUMANTranscription factor p65v rel reticuloendotheliosis viral oncogene homolog Av rel reticuloendotheliosis viral oncogene homolog A (avian)
      see all
  • Database links

      • Applications

      Our Abpromise guarantee covers the use of ab119664 in the following tested applications.

      The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

      Application Notes
      Sandwich ELISA sELISA. Tested in HeLa, THP1, Jurkat.

      PhosphoTracer NFkappaB p65 pSer536 + Total ELISA Kit images

      • Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Using the NFKappaB p65 assay kits, a significant stimulation of NFKappaB p65 phosphorylation at Ser536 is detected in HeLa cells treated with TNF alpha for 10 minutes compared with untreated cells, while no change in total NFKappaB p65 levels is observed.
      • Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Using the PhosphoTracer phospho-NFKappaB p65 assay, the amount of lysate required to detect NFKappaB p65 in TNF alpha-treated HeLa cell lysates was examined. Up to 50 µg of lysate/well was loaded into replicate wells of a PhosphoTracer microplate, and analysed for phospho-NFKappaB p65. Phospho-NFKappaB p65 was readily detected in less than 10 µg of lysate/well.
      • Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Sandwich ELISA - PhosphoTracer NFkappaB p65 (pS536) + total NFkappaB p65 ELISA kit (ab119664)
        Day 1: HeLa cells were seeded at 25K cells/well in a 96 well tissue culture plate in medium containing 10% FBS, and incubated overnight. Day 2: The media was removed and cells were stimulated with various concentrations of TNF aplha for 10 minutes. The media was removed, and the cells were lysed with 125 µL/well freshly prepared Lysis Mix, with shaking for 10 min. Lysates (50 µL) were transferred to replicate wells of an PhosphoTracer assay plate and analyzed for either phospho or total NFKappaB p65 using the standard PhosphoTracer protocol. Briefly, Antibody Mix specific for either phospho-NFKappaB p65, or Total NFKappaB p65 (50 µL/well), was added to the lysate, and the plate was incubated for 1 hr at room temp, with shaking. The plate was washed and Substrate Mix was added to the wells. The plate was covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.

      Protocols

      References for PhosphoTracer NFkappaB p65 pSer536 + Total ELISA Kit (ab119664)

      ab119664 has not yet been referenced specifically in any publications.

      Publishing research using ab119664 ? Please let us know so that we can cite the reference in this datasheet

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      We have your elisa kit.(ab119664 PhosphoTracer NFB p65 (pS536) + total NFB p65 ELISA Kit )

      Can I use to your kit for p65 activity from fresh atherosclerotic plaque ?

      how I prepared Cell lysed from atherosclerotic plaque?

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      Thank you for contacting us.

      Unfortunately, it seems that here is a misunderstanding: We have established this kit with a cell culture lysate of HeLa cells.

      From the email I can see that you are planning to use tissue culture lysate. ...

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      Could they (ab119664 and ab119674) use for western blot ?
      My customer want to detect NF kappa B by ELISA and western blot , which product should they use ?
      Thank you very much.

      Thank you for contacting us.

      Indeed, we can confirm that the antibodies provided in the mentioned PhosphoTracer ELISA kits can also be used for western blot. In fact, it would be great if you could let us know how the antibodies/kits work for y...

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