NFkB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit (Colorimetric) (ab207216)

Overview

  • Product name
    NFkB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit (Colorimetric)
  • Detection method
    Colorimetric
  • Sample type
    Cell Lysate, Nuclear Extracts
  • Assay type
    Semi-quantitative
  • Sensitivity
    < 500 ng/well
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Human
  • Product overview

    NFkB (p50, p52, p65, c-Rel, RelB) Transcription Factor Assay Kit (Colorimetric) (ab207216) is a high throughput assay to quantify activation of NFkB (p50, p52, p65, c-Rel, RelB) factors at the same time in one plate. 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 consensus binding site (5´ - GGGACTTTCC - 3´) has been immobilized onto a 96-well plate. Active NFkB present in nuclear or whole cell extracts specifically binds to the oligonucleotide. NFkB is detected by primary antibodies that recognizes an epitope on p50, p52, p65, c-Rel or RelB accessible only when NFkB is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout at OD 450 nm. This product detects human, mouse and rat NFkB p65 and RelB, human and mouse p50 and p52, and human c-Rel.


    Key performance and benefits:



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

    • Detection limit: < 0.5 µg nuclear extract/well.

    • Detection range: 0.2 – 5 µg nuclear 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. The DNA-binding protein complex recognizes a discrete nucleotide sequence (5´ - GGGACTTTCC - 3´) in the upstream region of a variety of cellular and viral response genes. 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. This domain is also a target of the IkB inhibitors, which include IkBα, IkBβ, IkBγ, Bcl-3, p105 and p100.

    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 (NFkB1/RelA) heterodimers and the p50 homodimers are the most common dimers found in the NFkB signaling pathway. 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. Proteolytic cleavage of p105 results in two proteins: p50, which has DNA-binding activity but no transactivation domain, and its antagonist, the inhibitory IkBγ 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.

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 2 x 96 tests
    10X Antibody Binding Buffer 2 x 2.2ml
    10X Wash Buffer 1 x 60ml
    96-well NFkB assay plate 2 units
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 2 x 11µl
    Binding Buffer 1 x 10ml
    Developing Solution 2 x 11ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl
    Herring sperm DNA (1 μg/μL) 1 x 100µl
    Lysis Buffer 1 x 10ml
    Mutated oligonucleotide (10 pmol/µL) 1 x 100µl
    NFkB c-Rel antibodies 1 x 11µl
    NFkB p50 antibodies 1 x 11µl
    NFkB p52 antibodies 1 x 11µl
    NFkB p65 antibody 1 x 11µl
    NFkB RelB antibodies 1 x 11µl
    Plate sealer 2 units
    Protease Inhibitor Cocktail 1 x 100µl
    Raji nuclear extract (2.5 µg/µL) 1 x 40µl
    Stop Solution 1 x 60ml
    Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl
  • Research areas
  • Relevance
    NFKB has been detected in numerous cell types that express cytokines, chemokines, growth factors, cell adhesion molecules, and some acute phase proteins in health and in various disease states. NFKB is activated by a wide variety of stimuli such as cytokines, oxidant-free radicals, inhaled particles, ultraviolet irradiation, and bacterial or viral products. Inappropriate activation of NF-kappa-B has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NF-kappa-B has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth.
  • Cellular localization
    Cytoplasmic and Nuclear. Note=Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B).
  • Alternative names
    • MGC131774
    • NFKB 3
    • NFKB3
    • Nuclear factor NF kappa B p65 subunit
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 p65
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 p65 (avian)
    • p65
    • RELA
    • Transcription factor p65
    • v rel avian reticuloendotheliosis viral oncogene homolog A
    • v rel reticuloendotheliosis viral oncogene homolog A
    see all
  • Database links

Associated products

Images

  • Nuclear extracts (10 µg/well) prepared a variety of cell lines were tested for activity of the different NFkB family members. Cell lines were untreated HeLa cells, HeLa cells treated with TNFα, untreated Jurkat cells, Jurkat cells treated with PMA and calcium ionophore (CI), and Raji cells. These results are provided for demonstration only.

Protocols

References

ab207216 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

There are currently no Customer reviews or Questions for ab207216.
Please use the links above to contact us or submit feedback about this product.

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

Sign up