NF-kappaB p65 Total PhosphoTracer ELISA Kit (ab119637)

Overview

  • Product nameNF-kappaB p65 Total PhosphoTracer ELISA KitSee all NF-kappaB p65 (Total) kits ...
  • Detection methodFluorescent
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts
  • Assay typeSemi-quantitative
  • Sensitivity
    10 µg protein/ml
  • Range
    10 µg protein/ml - 1000 µg protein/ml
  • Assay time
    2h 0m
  • 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 NF-kappaB p65 assay kits detect endogenous levels of NF-kappaB p65 (GenBank Accession NP_068810) in cellular lysates. Total NF-kappaB p65 assay kits detect NF-kappaB p65 irrespective of phosphorylation status.



    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 ELISAmore 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.
  • Information by UniProt
  • Alternative names
    • NF kappa B p65delta3
    • NFKB, p65 subunit
    • NFKB3
    • Nuclear factor kappa-B, subunit 3
    • Nuclear factor NF-kappa-B p65 subunit
    • Nuclear factor of kappa light chain gene enhancer in B cells3
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3
    • p65
    • relA
    • TF65_HUMAN
    • Transcription factor NFKB3
    • Transcription factor p65
    • V rel reticuloendotheliosis viral oncogene homolog A
    • V rel reticuloendotheliosis viral oncogene homolog A (avian)
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab119637 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
Sandwich ELISA Use at an assay dependent concentration. Tested in HeLa, THP1, Jurkat

NF-kappaB p65 Total PhosphoTracer ELISA Kit images

  • Detection of total NF-kappaB p65, using ab119637 on HeLa cells stimulated with various concentrations of TNF alpha.
  • Using the PhosphoTracer Total NF-kappaB p65 assay, the amount of lysate required to detect NF-kappaB p65 in TNFalpha-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 total NF-kappaB p65. Total NF-kappaB p65 was readily detected in less than 10 μg of lysate/well.
  • Using the NF-kappaB p65 assay kits, a significant stimulation of NF-kappaB p65 phosphorylation at Ser536 is detected in HeLa cells treated with TNFalpha for 10 minutes (+) compared with untreated cells (-), while no change in total NF-kappaB p65 levels is observed.
  • Using Western blot, a significant stimulation of NF-kappaB p65 phosphorylation at Ser536 is detected in HeLa cells treated with TNFalpha for 10 minutes (+) compared with untreated cells (-), while no change in total NF-kappaB p65 levels is observed.

Protocols

References for NF-kappaB p65 Total PhosphoTracer ELISA Kit (ab119637)

ab119637 has not yet been referenced specifically in any publications.

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We have not tested these kits in serum/plasma, however there is no particular reason the kits cannot be used. Of course, precautions should be taken when working with serum, including controlling for the many interfering factors that can occu...

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Thank you for contacting us and for your interest in our products.

In answer to your question, for longer incubations, the lysate/Ab mix incubation step is lengthened to increase sensitivity. It should be noted that this is a case-by-case rec...

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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 fo...

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