Overview

  • Product nameNF kappaB p65 ELISA Kit
  • Detection methodColorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    HeLa extract 6 2.8%
    Inter-assay
    Sample n Mean SD CV%
    HeLa extract 3 8.7%
  • Sample type
    Cell Lysate, Tissue Homogenate
  • Assay typeSemi-quantitative
  • Sensitivity
    < 5 µg/ml
  • Range
    6 µg/ml - 600 µg/ml
  • Assay time
    1h 30m
  • Assay durationOne step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    Abcam’s NF?B p65 in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of NF?B p65 protein in Human and mouse cells.

    The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Tested applicationsSuitable for: Sandwich 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 ab176648 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.

NF kappaB p65 ELISA Kit images

  • Example of a typical NFĸB p65 cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.

  • Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1 x Cell Extraction Buffer PTR. Data from duplicate measurements of NFĸB p65 (Total) are normalized and plotted.

  • Cell line analysis for Total NFĸB p65 from 200 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).

  • Induction of NFĸB p65 (pS536) phosphorylation in MCF-7 cells in response to TNFα treatment. HeLa cells were cultured in 96-well tissue culture plates, and treated (10 min) with a dose-range of TNFα before cell lysis. Data from quadruplicate measurements of NFĸB p65 (pS536) are plotted and compared against total NFĸB p65 protein levels. Comparative NFĸB p65 (pS536) and NFĸB p65 (Total) data also shown by Western Blot.

Protocols

References for NF kappaB p65 ELISA Kit (ab176648)

ab176648 has not yet been referenced specifically in any publications.

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