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    products/primary-antibodies/nf-kb-p65-antibody-ep2161y-ab76311.pdf

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Cell Biology Apoptosis Intracellular NFkB RelA (p65)
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

  • Datasheet
  • SDS
Reviews (1)Q&A (1)References (16)

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Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
  • Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
  • Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
  • Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
  • Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
  • Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP2161Y] to NF-kB p65
  • Suitable for: WB, ICC/IF, Flow Cyt (Intra)
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free

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Protein
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Recombinant Human NF-kB p65 protein (Tagged) (ab114150)
Primary
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Alexa Fluor® 488 Anti-NF-kB p65 antibody [EP2161Y] (ab204263)
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Anti-Acid phosphatase antibody [EPR21787] (ab235448)

View more associated products

Overview

  • Product name

    Anti-NF-kB p65 antibody [EP2161Y]
    See all NF-kB p65 primary antibodies
  • Description

    Rabbit monoclonal [EP2161Y] to NF-kB p65
  • Host species

    Rabbit
  • Specificity

    ab76311 detects both phosphorylated and non-phosphorylated versions of human NF-kB p65.
  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cyt (Intra)more details
    Unsuitable for: IHC-P or IP
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HeLa, Daudi and Jurkat cell lysates. Flow Cyt (intra): HeLa and HT-29 cells. ICC: HT-29 cells
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.5% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP2161Y
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • NFkB
    • RelA (p65)
    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Alzheimer's disease
    • Other
    • Microbiology
    • Interspecies Interaction
    • Host Virus Interaction
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFkB Pathway
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • NFkB pathway
    • Cancer
    • Signal transduction
    • Nuclear signaling
    • NFkB pathway
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Inflammatory mediators
    • Microbiology
    • Interspecies Interaction
    • Host Immune Response
    • Metabolism
    • Types of disease
    • Obesity
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • NFkB
    • RelA (p65)

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-NF-kB p65 antibody [EP2161Y] (ab204263)
    • Alexa Fluor® 647 Anti-NF-kB p65 antibody [EP2161Y] (ab204649)
    • Anti-NF-kB p65 antibody [EP2161Y] - BSA and Azide free (ab246347)
  • Assay kits

    • NFkB p65 Transcription Factor Assay Kit (ab133112)
  • Compatible Secondaries

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Jurkat cytoplasmic extract lysate (ab14842)
  • Recombinant Protein

    • Recombinant Human NF-kB p65 protein (Tagged) (ab114150)
  • Related Products

    • NF kappaB p65 ELISA Kit (ab176648)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab76311 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
WB (1)
1/1000 - 1/5000. Detects a band of approximately 70 kDa (predicted molecular weight: 65 kDa).
ICC/IF
1/100 - 1/500.
Flow Cyt (Intra)
1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

Notes
WB
1/1000 - 1/5000. Detects a band of approximately 70 kDa (predicted molecular weight: 65 kDa).
ICC/IF
1/100 - 1/500.
Flow Cyt (Intra)
1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

Application notes
Is unsuitable for IHC-P or IP.

Target

  • 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.
  • Target information above from: UniProt accession Q04206 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 5970 Human
    • Omim: 164014 Human
    • SwissProt: Q04206 Human
    • Unigene: 502875 Human
    • Alternative names

      • Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
      • MGC131774 antibody
      • NF kappa B p65delta3 antibody
      • nfkappabp65 antibody
      • NFkB p65 antibody
      • NFKB3 antibody
      • Nuclear factor kappaB 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 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 NFKB3 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
      see all

    Images

    • Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: NF-κB p65 knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: A431 cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab76311 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.


      ab76311 was shown to specifically react with NF-κB p65 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when NF-κB p65 knockout samples were used. Wild-type and NF-κB p65 knockout samples were subjected to SDS-PAGE. ab76311 (NF-κB p65) and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

      Immunocytochemistry/Immunofluorescence analysis HT-29 (human colorectal adenocarcinoma) labelling NF-kB p65 with purified ab76311 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

      Control: PBS only

    • Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

      Flow Cytometry analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab76311 at 1/1000 dilution (1 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

    • Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Western blot - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      All lanes : Anti-NF-kB p65 antibody [EP2161Y] (ab76311) at 1/20000 dilution

      Lane 1 : HeLa cell lysate
      Lane 2 : Daudi cell lysate
      Lane 3 : Jurkat cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 65 kDa
      Observed band size: 70 kDa why is the actual band size different from the predicted?

    • Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Flow Cytometry (Intracellular) - Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

      Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab76311 at 1/1000 dilution (1 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

    • Anti-NF-kB p65 antibody [EP2161Y] (ab76311)
      Anti-NF-kB p65 antibody [EP2161Y] (ab76311)

    Protocols

    • Flow cytometry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (16)

    Publishing research using ab76311? Please let us know so that we can cite the reference in this datasheet.

    ab76311 has been referenced in 16 publications.

    • Li Q  et al. Carminic acid supplementation protects against fructose-induced kidney injury mainly through suppressing inflammation and oxidative stress via improving Nrf-2 signaling. Aging (Albany NY) 13:10326-10353 (2021). PubMed: 33819919
    • Liu H  et al. MicroRNA-421 attenuates macrophage-mediated inflammation by inhibiting PDCD4 in vitro. Mol Med Rep 24:N/A (2021). PubMed: 34036390
    • Tuasha N  et al. Solvent fractions of selected Ethiopian medicinal plants used in traditional breast cancer treatment inhibit cancer stem cells in a breast cancer cell line. BMC Complement Med Ther 20:366 (2020). PubMed: 33238963
    • Deji QZ  et al. Cross-talk between microRNA-let7c and transforming growth factor-ß2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells. Int J Ophthalmol 13:693-700 (2020). PubMed: 32420214
    • Hsueh YS  et al. Nuclear KIT induces a NFKBIB-RELA-KIT autoregulatory loop in imatinib-resistant gastrointestinal stromal tumors. Oncogene 38:6550-6565 (2019). PubMed: 31363162
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-2 of 2 Abreviews or Q&A

    Western blot abreview for Anti-NF-kB p65 antibody [EP2161Y]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (neural stem cell)
    Gel Running Conditions
    Reduced Denaturing (4-12%)
    Loading amount
    40 µg
    Specification
    neural stem cell
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jul 03 2018

    Question

    Do the following antibodies recognize an extracellular epitope or do you have to follow an intracellular protocol for flow? ab238, 80392, 40752, 32041, 76429, 32135, 76311, 10684, 32536, 32360

    Read More

    Abcam community

    Verified customer

    Asked on Jan 12 2011

    Answer

    Thank you for contacting us. Flow cytometry protocols are recommended below. For most of the antibodies we are not able to provide specific epitope information but it seems most targets are intracellular. Hence not on the membrane and cells need fixation prior to staining. ab238- intracellular target. Intracellular protocol is recommended for flow cytometry. ab10684 -intracellular target. Intracellular protocol is recommended for flow cytometry. ab32041- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32135- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32536- intracellular target. Intracellular protocol is recommended for flow cytometry. ab32360- intracellular target. Intracellular protocol is recommended for flow cytometry. ab40752- intracellular target. Intracellular protocol is recommended for flow cytometry. ab76311- intracellular target. Intracellular protocol is recommended for flow cytometry. ab76429- intracellular target. Intracellular protocol is recommended for flow cytometry. ab80392- Bcl10 antibody [151] (Phycoerythrin) - Does not need a secondary detection antibody. Intracellular target. Intracellular protocol is recommended for flow cytometry. Our protocols are found on https://www.abcam.com/protocols I have attached a protocol for intracellular targets from one of our collaborators (this protocol is also inluded under the link) I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Abcam Scientific Support

    Answered on Jan 12 2011

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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