NFkB p65 Transcription Factor Assay Kit (ab133112)

Overview

  • Product nameNFkB p65 Transcription Factor Assay Kit
    See all NF-kB p65 kits
  • Sample type
    Cell culture extracts, Nuclear Extracts
  • Assay typeSemi-quantitative
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Abcam's NFkB p65 Transcription Factor Assay Kit (ab133112) is a non-radioactive, sensitive method for detecting specific transcription factor DNA binding activity in nuclear extracts.

    A 96-well enzyme-linked immunosorbent assay (ELISA) replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA). A specific double stranded DNA (dsDNA) sequence containing the NFkB response element is immobilized onto the bottom of wells of a 96-well plate. NFkB contained in a nuclear extract, binds specifically to the NFkB response element. NFkB (p65) is detected by addition of specific primary antibody directed against NFkB (p65). A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.

  • Tested applicationsSuitable for: Functional Studiesmore details

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
    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A
    • MGC131774
    • NF kappa B p65delta3
    • NFKB3
    • 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 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

Applications

Our Abpromise guarantee covers the use of ab133112 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
Functional Studies Use at an assay dependent concentration.

It does not cross-react with NFkB (p50) transcription factor.

NFkB p65 Transcription Factor Assay Kit images

  • Jurkat cells were treated with PMA and ionomycin (+). Nuclear lysates were extracted (ab113474) and 40 uL, corresponding to 4e6 cells, were tested in duplicates (+/- SD).

  • Titration of positive control with or without inhibitor, background signal subtracted (duplicates; +/- SD).

  • Assay of cell lysates isolated from stimulated (20 ng/ml TNF alpha for 30 minutes) and nonstimulated HeLa cells demonstrating NFkB (p65) activity.

Protocols

References for NFkB p65 Transcription Factor Assay Kit (ab133112)

ab133112 has not yet been referenced specifically in any publications.

Product Wall

For tissue samples, in place of steps 1 and 2 in the kit protocol part B. Purification of Cellular Nuclear Extracts, we recommend the following procedure with fresh tissue:

1. Weigh a fresh tissue sample and cut into very small pieces using ...

Read More
The samples came from young male mice frozen subcutaneous tissue. We got the nuclear extraction by Thermo nuclear extraction kit. The nuclear extraction protein concentration were not high. We removed the lipid layer after centrifuge in 4 degree.
The background and positive control OD value were very positive. The sample OD values were a little bit low. The reason may be that the tissue was not fresh the nuclear extraction kit may contain some salt that may interfere with the NFkB kit the nuclear protein concentration was too low.
We proceed the experiment as the protocol.
Overall the kit works well.
Username

Abcam user community

Verified customer

Submitted May 15 2015

We recommend using 10 uL of sample per well. The nuclear extraction protocol in the kit results in the production of 100 uL of sample with approximately 50 ug of nuclear protein from 10^7 cells. If you are using a significantly smaller number of cells ...

Read More


1- what is the benefit and meaning of Non-specific Binding Wells and Specific Competitor dsDNA Wells?
Answer: The non-specific binding wells will provide background data. The Specific competitor dsDNA wells involves addition of soluble p65 sp...

Read More

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