Overview

  • Product nameAnti-NF-kB p65 antibody - ChIP Grade
    See all NF-kB p65 primary antibodies
  • Description
    Rabbit polyclonal to NF-kB p65 - ChIP Grade
  • SpecificityReacts with NF-kB p65
  • Tested applicationsChIP, ICC, ChIP/Chip, EMSA, IP, IHC-P, WB, IHC-Fr, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human NF-kB p65 aa 532-551 (C terminal).
    Sequence:

    EDFSSIADMDFSALLSQISS


    Database link: Q04206
    (Peptide available as ab8005)

  • Positive control
    • K-562, NIH/3T3, Hela and A431 cell lysates human DLBCL and human inflammatory skin biopsy

Applications

Our Abpromise guarantee covers the use of ab7970 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
ChIP Use at an assay dependent dilution.
ICC Use at an assay dependent concentration.
ChIP/Chip Use at an assay dependent concentration.
EMSA Use a concentration of 1 µg/ml.
IP Use at an assay dependent dilution.
IHC-P 1/1000 - 1/4000. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
WB 1/400 - 1/1000. Detects a band of approximately 64 kDa (predicted molecular weight: 69 kDa).Can be blocked with NF-kB p65 peptide (ab8005).
IHC-Fr 1/100. PubMed: 17003500
ICC/IF Use at an assay dependent dilution. PubMed: 16818736
Flow Cyt Use at an assay dependent concentration.

Target

  • 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
  • Database links
  • Alternative names
    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
    • MGC131774 antibody
    • NF kappa B p65delta3 antibody
    • NFKB 3 antibody
    • NFKB3 antibody
    • Nuclear Factor NF Kappa B p65 Subunit antibody
    • Nuclear factor NF-kappa-B p65 subunit antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 antibody
    • Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B Cells antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 antibody
    • OTTHUMP00000233473 antibody
    • OTTHUMP00000233474 antibody
    • OTTHUMP00000233475 antibody
    • OTTHUMP00000233476 antibody
    • OTTHUMP00000233900 antibody
    • p65 antibody
    • p65 NF kappaB antibody
    • p65 NFkB antibody
    • RELA antibody
    • TF65_HUMAN antibody
    • Transcription Factor p65 antibody
    • v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)) antibody
    • V Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A antibody
    • v rel reticuloendotheliosis viral oncogene homolog A (avian) antibody
    • V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65 antibody
    • v-rel reticuloendotheliosis viral oncogene homolog A antibody
    see all

Anti-NF-kB p65 antibody - ChIP Grade images

  • ab7970 used undiluted for ChIP analysis of mouse primary T cell nuclear lysate, incubated for 16 hours at 4°C. Cross-linking (X-ChIP) using formaldehyde for 10 minutes.
    Detection step: Real-time PCR.
    Negative control: Rabbit IgG.
    Results showed the dramatic enrichment of p65 binding upon P+I stimulation in IL-4 promoter region and also IL-2 promoter region.

    See Abreview

  • ICC/IF image of ab7970 stained HEK293 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7970, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab7970 staining NF-kB p65 in Human AGS cells by ICC/IF (Immunocytochemistry/immunoflurescence). Cells were fixed with formaldehyde, permeabilzed with 0.025% Trton X-100 in TBS and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/100) for 1 hour at 25°C. An Alexa Fluor®568-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • All lanes : Anti-NF-kB p65 antibody - ChIP Grade (ab7970)

    Lane 1 : A431 whole cell lysate
    Lane 2 : Hela cell lysate


    Performed under reducing conditions.

    Predicted band size : 69 kDa
    Observed band size : 64 kDa (why is the actual band size different from the predicted?)

    Western blotting starting dilution: 1/400-1/1000.

  • Anti-NF-kB p65 antibody - ChIP Grade (ab7970) at 1/1000 dilution + Lysate prepared from mouse lung tissue at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/20000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 69 kDa
    Observed band size : 69 kDa


    Exposure time : 15 seconds

    This image is a courtesy of Anonymous Abreview

    See Abreview

  • Immunoperoxidase staining of formalin-fixed, paraffin-embedded human DLBCL showing nuclear expression of NF-kB p65 in the tumor cells.

  • ab7970 staining NF-kB p65 in Human stomach tissue sections by IHC-Fr (Immunohistochemistry - Froxen sections). Tissue samples were fixed with acetone and blocked with 5% serum for 1 hour at 25°C. Samples were incubated with primary antibody 1/1000 in blocking buffer for 1 hour at 25°C. An undiluted HRP-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody.

    See Abreview

  • ab7970 staining NF-kB p65 in mouse lung tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent xylene fixation before heat mediated antigen retrieval in citrate and then blocking with 1.5% serum was performed for 30 minutes at 37°C. The primary antibody was used diluted 1/500 and incubated with sample for 16 hours at 4°C. A Biotin conjugated polyclonal to rabbit IgG was used undiluted as secondary antibody. ABC staining system was used.

    See Abreview

References for Anti-NF-kB p65 antibody - ChIP Grade (ab7970)

This product has been referenced in:
  • Laprairie RB  et al. The cytokine and endocannabinoid systems are co-regulated by NF-?B p65/RelA in cell culture and transgenic mouse models of Huntington's disease and in striatal tissue from Huntington's disease patients. J Neuroimmunol 267:61-72 (2014). Mouse, Human . Read more (PubMed: 24360910) »
  • DeDiego ML  et al. Inhibition of NF-?B-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival. J Virol 88:913-24 (2014). WB ; Mouse . Read more (PubMed: 24198408) »

See all 68 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Non-reduced Denaturing (8)
Sample Human Cell lysate - whole cell (MEFs, U2OS, HEK 293 and Fibroblasts)
Specification MEFs, U2OS, HEK 293 and Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Sep 23 2014

Application ChIP
Detection step Real-time PCR
Sample Rat Cell lysate - whole cell (h9c2)
Specification h9c2
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde Solution
Username

Abcam user community

Verified customer

Submitted Oct 17 2013

ChIP

Excellent
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (mouse microglia cells)
Specification mouse microglia cells
Negative control 1. untreated sample 2. Rabbit IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Positive control LPS treated sample
Username

Abcam user community

Verified customer

Submitted Jun 04 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - whole cell (hek293)
Total protein in input 100 µg
Specification hek293
Immuno-precipitation step Other - dynabeads IgG
Username

Abcam user community

Verified customer

Submitted May 17 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (hek293)
Loading amount 30 µg
Specification hek293
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
Username

Abcam user community

Verified customer

Submitted May 17 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (PBMC)
Loading amount 30 µg
Specification PBMC
Gel Running Conditions Reduced Denaturing (10%)
Blocking step Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted May 14 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Rat Cell lysate - whole cell (h9c2 cell)
Loading amount 30 µg
Specification h9c2 cell
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
Username

Abcam user community

Verified customer

Submitted Apr 08 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Rat Tissue lysate - whole (h9c2 cell)
Total protein in input 200 µg
Specification h9c2 cell
Immuno-precipitation step Other - Dynabeads
Username

Abcam user community

Verified customer

Submitted Apr 08 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Rat Cell (h9c2 cell)
Specification h9c2 cell
Fixative Paraformaldehyde
Permeabilization Yes - 1% triton x100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
Username

Abcam user community

Verified customer

Submitted Apr 05 2013

Thank you for contacting us.

IHC-P has not been tested with this antibody so this application so this application is not covered under Abpromise. However if customer is using this antibody in IHC-P then we can provide Abtrial discount. Please ...

Read More

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