Overview

  • Product name
    Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade
    See all NF-kB p65 primary antibodies
  • Description
    Rabbit polyclonal to NF-kB p65 (acetyl K310) - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, Dot blot, ICC, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human NF-kB p65 aa 300-400 (internal sequence) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab20612)

  • Positive control
    • This antibody gave a positive signal in Rat lung tissue lysate.

Applications

Our Abpromise guarantee covers the use of ab19870 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 Use a concentration of 2.5 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa). Collaborator data suggests that immunoprecipitation of this antibody prior to Western blotting is required to obtain the best results (see images)
IP Use a concentration of 2.5 µg/ml.
Dot blot Use at an assay dependent dilution.
ICC Use at an assay dependent dilution. In ICC/IF ab19870 recognizes various acetylated nuclear protein(s), as the signal is also observed in control cells; the signal in ICC is HDACi-dependent.
ChIP Use at an assay dependent concentration. PubMed: 22249179

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.
  • Information by UniProt
  • Database links
  • Alternative names
    • Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
    • MGC131774 antibody
    • NF kappa B p65delta3 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 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
    see all

Images

  • Lanes 1-2 : Coomassie stain
    Lanes 3-4 : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1/500 dilution

    Lanes 1 & 3 : Acetylated p65 protein
    Lanes 2 & 4 : K310 mutant protein

    Lysates/proteins at 2 µg per lane.

    Secondary
    Lanes 3-4 : IRDye® 800CW Goat anti-Rabbit IgG (H + L) at 1/15000 dilution

    Predicted band size: 65 kDa



    Observed band size: 75kDa

    The p65 band runs higher in this blot because the protein contains a myc-tag.

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1/1000 dilution

    Lane 1 : HCT116 whole cell lysate treated with DMSO for 24 hrs (control)
    Lane 2 : HCT116 whole cell lysate treated with 2 µM SAHA for 24 hrs
    Lane 3 : MEF whole cell lysate treated with DMSO for 24 hrs (control)
    Lane 4 : MEF whole cell lysate treated with 2 µM SAHA for 24 hrs

    Lysates/proteins at 60 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 65 kDa
    Observed band size: 65 kDa
    Additional bands at: 140 kDa (possible non-specific binding), 15 kDa (possible non-specific binding), 40 kDa (possible non-specific binding), 45 kDa (possible non-specific binding), 90 kDa (possible non-specific binding)


    Exposure time: 2 minutes

    See Abreview

  • Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3h at room temperature. Exposure time: 75 min normal ECL. This Dot blot demonstrates that ab19870 recognized upto 10ng of purified peptide on a PVDF membrane.

  • Western Blot with ab19870 after p65 Immunoprecipitation: rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870; 2.5µg/ml) in 1% non-fat milk TBS-T incubated for 3 hours at room temperature. Exposure time: 1 min normal ECL. Tested samples: nuclear extracts (180 µg) of immortalized p65-/- mouse cells, complemented with the empty vector (pRRL), wild-type p65 (Wt) and non-acetylatable K310 (K310R). The samples tested were treated with deacetylase inhibitors HDACi (TSA + Nicotinamide) and TNF-alpha. The samples were immunoprecipitated with 2µg of alpha-p65 and subsequently analysed by Western blot with Rabbit polyclonal to NF-kB p65 (acetyl K310) (ab19870). Predicted band size = 65kDa, Observed band size = 75kDa. The p65 band runs higher in this SDS-PAGE blot as it contains a myc-tag. 

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 2.5 µg/ml

    Lane 1 : pRRL untreated
    Lane 2 : pRRL HDACi
    Lane 3 : pRRL HDACi + TNF
    Lane 4 : Wt untreated
    Lane 5 : Wt HDACi
    Lane 6 : Wt HDACi + phorbol myristate acetate
    Lane 7 : K310R untreated
    Lane 8 : K310R HDACi
    Lane 9 : K310R HDACi + phorbol myristate acetate

    Lysates/proteins at 75 µg per lane.

    Developed using the ECL technique.

    Predicted band size: 65 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 hour


    ab19870 recognizes Rabbit polyclonal to NF-kB p65 (acetyl K310) specifically at ~75kDa (indicated by the arrow) is this SDS-PAGE blot. The p65 band runs higher than 65kDa in this SDS-PAGE blot as it contains a myc-tag. We are sure that the band at ~75kDa is p65 since p65 specific antibodies detect the same band in IP and WB and there is no signal in the p65 knock-out cell line with ab19870. A number of additonal bands are recognized by ab19870 when tested with endogenous p65 from whole cell extracts, we do not know the identity of these bands.

    Tested samples: nuclear extracts (75µg) of immortalized p65-/- mouse cells, complemented with the empty ve

  • Anti-NF-kB p65 (acetyl K310) antibody - ChIP Grade (ab19870) at 1 µg/ml + Lung (Rat) Tissue Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 65 kDa
    Observed band size: 72 kDa why is the actual band size different from the predicted?
    Additional bands at: 15 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes

References

This product has been referenced in:
  • Sanajou D  et al. Reduction of renal tubular injury with a RAGE inhibitor FPS-ZM1, valsartan and their combination in streptozotocin-induced diabetes in the rat. Eur J Pharmacol 842:40-48 (2019). Read more (PubMed: 30393200) »
  • Wang X  et al. Astragaloside IV inhibits glucose-induced epithelial-mesenchymal transition of podocytes through autophagy enhancement via the SIRT-NF-?B p65 axis. Sci Rep 9:323 (2019). Read more (PubMed: 30674969) »
See all 45 Publications for this product

Customer reviews and Q&As

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1-6 of 6 Abreviews

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Tissue lysate - whole (Placenta)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
15 µg
Specification
Placenta
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Jeremiah Gaudet

Verified customer

Submitted Aug 23 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Mouse colon cells)
Permeabilization
No
Specification
Mouse colon cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Methanol

Abcam user community

Verified customer

Submitted Jan 25 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Bladder)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: PH6 Citrate Buffer
Permeabilization
No
Specification
Bladder
Blocking step
HIHS as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Paraformaldehyde

Mr. Spenser Souza

Verified customer

Submitted May 12 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Testis)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer ph6 20 min at 97ºC in a PT link
Permeabilization
No
Specification
Testis
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 24 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
100000 cells
Gel Running Conditions
Reduced Denaturing (12 %)
Sample
Human Cell lysate - whole cell (breast)
Specification
breast
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 20 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HCT116 colon cancer cell line and MEF cell line)
Loading amount
60 µg
Specification
HCT116 colon cancer cell line and MEF cell line
Treatment
DMSO control and 2 µM SAHA for 24 hrs
Gel Running Conditions
Reduced Denaturing (13%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Mr. Christian Marx

Verified customer

Submitted Feb 11 2013

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

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