Overview

  • Product nameAnti-NF-kB p65 antibodySee all NF-kB p65 primary antibodies ...
  • Description
    Rabbit polyclonal to NF-kB p65
  • Tested applicationsIHC-FoFr, ICC/IF, IHC-P, WB, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Human, Indian Muntjac, Other
  • Immunogen

    Synthetic peptide corresponding to Human NF-kB p65 aa 500 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab16636)

  • Positive control
    • This antibody gave a positive signal in the following lysates: Mouse Spleen Tissue, HeLa Whole Cell, A431 Whole Cell

Properties

Applications

Our Abpromise guarantee covers the use of ab16502 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
IHC-FoFr Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 0.5 µg/ml. Detects a band of approximately 64 kDa (predicted molecular weight: 60 kDa).Can be blocked with Human NF-kB p65 peptide (ab16636).
IP Use at an assay dependent concentration.
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 images

  • IHC image of NF-kB p65 staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16502, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  • Anti-NF-kB p65 antibody (ab16502) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 50000 mg/ml
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 60 kDa
    Observed band size : 64 kDa (why is the actual band size different from the predicted?)


    Exposure time : 4 minutes

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16502 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • All lanes : Anti-NF-kB p65 antibody (ab16502) at 1 µg/ml

    Lane 1 : Spleen (Mouse) Tissue Lysate
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 60 kDa
    Observed band size : 64 kDa (why is the actual band size different from the predicted?)


    Exposure time : 8 minutes
  • NF-kB p65 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to NFkB p65 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16502.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 68kDa: NFkB p65

  • ab16502 stining the nuclei of the cardiac cells in rat tissue. The tissues were fixed (animals perfused fixed) with 4% PFA and later postfixed overnight in the same fixative. They were cryoprotected in 30% sucrose and cut using a cryostat.

    See Abreview

  • ICC/IF image of ab16502 stained MCF7 cells. The cells were 4% PFA fixed (10 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 (ab16502, 1µg/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.



  • Performed under reducing conditions.

    Predicted band size : 60 kDa


  • Performed under reducing conditions.

    Predicted band size : 60 kDa
  • ab16502 at a 1/500 dilution staining Asynchronous and paraformaldehyde-fixed (4%) HeLa cells by immunocytochemistry. The antibody was incubated with the cells 30 minutes and then detected using a Cy3 conjugated Goat Anti-Mouse IgG (H+L) antibody.

    This image is courtesy of an Abreview by Kirk McManus submitted on 27 February 2006.

    See Abreview

  • ICC/IF image of ab16502 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16502, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 phalloidin was used to label F-actin (red).

References for Anti-NF-kB p65 antibody (ab16502)

This product has been referenced in:
  • Marongiu F  et al. Clearance of senescent hepatocytes in a neoplastic-prone microenvironment delays the emergence of hepatocellular carcinoma. Aging (Albany NY) 6:26-34 (2014). WB ; Rat . Read more (PubMed: 24464501) »
  • Sheppard PW  et al. Overexpression of heat shock protein 72 attenuates NF-?B activation using a combination of regulatory mechanisms in microglia. PLoS Comput Biol 10:e1003471 (2014). ICC/IF ; Mouse . Read more (PubMed: 24516376) »

See all 17 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 45 minute(s) · Concentration: 2% · Temperature: RT°C
Sample Human Cell (LS174t)
Specification LS174t
Permeabilization No
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 12 2013

Application Flow Cytometry
Fixation Formaldehyde
Sample Human Cell (Whole blood)
Specification Whole blood
Gating Strategy CD14 positive cells were gated for analysis
Permeabilization Yes - 0.1% Triton X-100
Username

Abcam user community

Verified customer

Submitted Dec 12 2013

Application Immunocytochemistry
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 3% · Temperature: RT°C
Sample Mouse Cultured Cells (RAW cell)
Specification RAW cell
Permeabilization Yes - 3% BSA in PBS with 0.1% Triton X-100
Fixative Paraformaldehyde
Username

Dr. Mukesh Jaiswal

Verified customer

Submitted Nov 13 2013

Application Immunohistochemistry (Frozen sections)
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3µg/mL · Temperature: RT°C
Sample Mouse Tissue sections (Uterus)
Specification Uterus
Permeabilization No
Fixative Paraformaldehyde
Username

Dr. Mukesh Jaiswal

Verified customer

Submitted Nov 13 2013

Merci de nous avoir contactés.


Nous avons 52 références anticorps anti-NFkB p65 dont certaines sont spécifiques de sites phosphorylés.

Je n'ai malheureusement pas trouvé d'information sur...

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Thank you for contacting us with your customer's questions.

At this time we have not tested any of our NFkappaB or IkappaB antibodies with zebrafish samples, so we do not know for sure which ones will cross-react. I have looked into the situa...

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Thank you for contacting us.

Your customers results are very interesting and it is indeed very possible that they have been detecting thedimeric form. This would certainly be supported by this band of ˜130 kDa only being present in the sample...

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La garantie Abpromise permet le remplacement par une autre référence même si le prix unitaire est différent, j'ai donc procédé à l'envoi d'une unité de ab16502. Le numéro de commande de...

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DISCOUNT CODE: *** Expiration date: April 19th, 2012 I am very pleased to hear you would like to accept our offer and test ab28856 in sheep. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit...

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Thank you for calling Abcam earlier. Please find attached the ICC/IF protocols that we talked about and below is the list of products that we discussed. ab7481, Goat Serum ab16502, Anti-NFkB p65 antibody ab31481, Anti-NFkB p65 antibody If there is anyt...

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