Overview

  • Product name

    NFkB p65 Transcription Factor Assay Kit (Chemiluminescent)
    See all NF-kB p65 kits
  • Detection method

    Luminescent
  • Sample type

    Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Sensitivity

    < 400 ng/well
  • Assay time

    3h 30m
  • Species reactivity


    Predicted to work with: Mouse, Rat, Human
  • Product overview

    NFkB p65 Transcription Factor Assay Kit (Chemiluminescent) (ab207221) is a high throughput assay to quantify NFkB p65 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.


    A specific double stranded DNA sequence containing the NFkB p65 consensus binding site (5’ – GGGACTTTCC – 3’) has been immobilized onto a 96-well plate. Active NFkB p65 present in nuclear specifically binds to the oligonucleotide. NFkB p65 is detected by a primary antibody that recognizes an epitope of NFkB p65 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides a sensitive chemiluminescent readout that can be quantified using a luminometer or CCD camera system. This product detects human, mouse and rat NFkB p65.


    Key performance and benefits:



    • Assay time: 3.5 hours (cell extracts preparation not included).

    • Detection limit: < 40 ng nuclear extract/well.

    • Detection range: 0.039 – 2.5 µg nuclear cell extract/well.

  • Notes

    The transcription factor NFkB is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses. NFkB is composed of homo- and heterodimeric complexes of members of the Rel (NFkB) family. There are five subunits of the NFkB family in mammals: p50, p65 (RelA), c-Rel, p52 and RelB. These proteins share a conserved 300 amino acid sequence in the N-terminal region, known as the Rel homology domain, that mediates DNA binding, protein dimerization and nuclear localization. Various dimer combinations of the NFkB subunits have distinct DNA binding specificities and may serve to activate specific sets of genes such as adhesion molecules, immunoreceptors and cytokines. The p50/p65 heterodimers are some of the most common dimers found in the NFkB signaling pathway. RelA (p65) heterodimers with both p50 and p52 participate in target gene transactivation.

    In the majority of cells, NFkB exists in an inactive form in the cytoplasm, bound to the inhibitory IkB proteins. Treatment of cells with various inducers results in the phosphorylation, ubiquitination and subsequent degradation of IkB proteins (For studying the phosphorylation state of IkBa. Proteolytic cleavage of p105 results in two proteins: p50, which has DNA-binding activity but no transactivation domain, and its antagonist, the inhibitory IkBg protein. This results in the release of NFkB dimers, which subsequently translocate to the nucleus, where they activate appropriate target genes. NFkB can be activated by a number of stimuli, including components of bacterial cell walls, such as lipopolysaccharide, or inflammatory cytokines, such as TNF-α or IL-1β.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well NFkB chemi assay plate 1 x 96 tests 5 x 96 tests
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    Chemiluminescent Reagent 1 x 2ml 1 x 10ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    Herring sperm DNA (1 μg/μL) 1 x 100µl 1 x 500µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    Mutated oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
    NFkB p65 antibody 1 x 11µl 1 x 55µl
    Plate sealer 1 unit 1 x 5 units
    Positive control nuclear extract 1 x 40µl 1 x 200µl
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Reaction Buffer 1 x 4ml 1 x 20ml
    Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
  • Research areas

  • 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
  • Alternative names

    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A
    • MGC131774
    • NF kappa B p65delta3
    • nfkappabp65
    • NFkB p65
    • NFKB3
    • Nuclear factor kappaB
    • Nuclear Factor NF Kappa B p65 Subunit
    • Nuclear factor NF-kappa-B p65 subunit
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3
    • OTTHUMP00000233473
    • OTTHUMP00000233474
    • OTTHUMP00000233475
    • OTTHUMP00000233476
    • OTTHUMP00000233900
    • p65
    • p65 NF kappaB
    • p65 NFkB
    • relA
    • TF65_HUMAN
    • Transcription factor NFKB3
    • Transcription factor p65
    • V rel avian reticuloendotheliosis viral oncogene homolog A
    • v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65))
    • v rel reticuloendotheliosis viral oncogene homolog A (avian)
    • V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65
    see all
  • Database links

Protocols

References

ab207221 has not yet been referenced specifically in any publications.

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