概述

  • 产品名称

    Anti-NF-kB p65抗体
    参阅全部 NF-kB p65 一抗
  • 描述

    兔多克隆抗体to NF-kB p65
  • 宿主

    Rabbit
  • 经测试应用

    适用于: IHC-FoFr, ICC/IF, IHC-P, WB, IP, Flow Cytmore details
  • 种属反应性

    与反应: Mouse, Rat, Chicken, Human, Indian muntjac, Heterocephalus glaber
  • 免疫原

    Synthetic peptide corresponding to Human NF-kB p65 aa 500 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab16636)

  • 阳性对照

    • This antibody gave a positive signal in the following lysates: Mouse Spleen Tissue, HeLa Whole Cell, A431 Whole Cell WB: Human fetal brain, kidney and lung tissue lysates.
  • 常规说明

      

性能

应用

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.

应用 Ab评论 说明
IHC-FoFr Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 - 5 µg/ml.
IHC-P Use a concentration of 1 - 5 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).

Abcam recommends using milk as the blocking agent.

IP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

靶标

  • 功能

    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.
  • 序列相似性

    Contains 1 RHD (Rel-like) domain.
  • 结构域

    the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
  • 翻译后修饰

    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'.
  • 细胞定位

    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
  • 数据库链接

  • 别名

    • 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

图片

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: NFκB 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 - ab16502 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.


    ab16502 was shown to react with NFκB p65 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when NFκB p65 knockout samples were used. Wild-type and NFκB p65 knockout samples were subjected to SDS-PAGE. ab16502 (NFκB 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 1/10 000 dilution for 1 hour at room temperature before imaging.

  • 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.

  • All lanes : Anti-NF-kB p65 antibody (ab16502) at 1/1000 dilution

    Lane 1 : Human fetal brain lysates
    Lane 2 : Human fetal kidney lysates
    Lane 3 : Human fetal lung lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 60 kDa
    Observed band size: 65 kDa
    why is the actual band size different from the predicted?



    Blocking/Diluting buffer and concentration: 5% NFDM/TBST

    Exposure Time: 15 seconds for Lane1 and 3 seconds for Lanes 2 and 3

    Normal brain might express low level of p65 (PMID: 21479220)

  • 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).

  • 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 Anti-Rabbit HRP (IgG light chain) (ab99697).
    Band: 68kDa: NFkB p65

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of VillinCre;Dclk1f/f mouse colon tissue sections labeling NF-kB p65 with ab16502 (brown). Alcian blue was used for counterstaining.Heat-induced epitope retrieval was performed on 4-μm formalin-fixed paraffin-embedded sections by utilizing a pressurized Decloaking Chamber in citrate buffer (pH 6.0) at 99°C for 18 min. For brightfield microscopy, slides were exposed to peroxidase blocking solution prior to the addition of primary antibody (ab16502). After incubation with primary antibody overnight at 4°C, the slides were incubated in peroxidase-conjugated polymer.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Dclk1f/f mouse colon tissue sections labeling NF-kB p65 with ab16502 (brown). Alcian blue was used for counterstaining.Heat-induced epitope retrieval was performed on 4-μm formalin-fixed paraffin-embedded sections by utilizing a pressurized Decloaking Chamber in citrate buffer (pH 6.0) at 99°C for 18 min. For brightfield microscopy, slides were exposed to peroxidase blocking solution prior to the addition of primary antibody (ab16502). After incubation with primary antibody overnight at 4°C, the slides were incubated in peroxidase-conjugated polymer.

     

  • 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 goat anti-rabbit DyLight® 488 (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.

  • 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
    All lanes : 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
  • ab16502 staining NF-kB p65 in murine peritoneal tumour cells by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 1% BSA for 60 minutes at room temperature. Samples were incubated with primary antibody (1/1000) for 2 hours. An undiluted Alexa Flour®647-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

  • 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

  • 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

  • All lanes : Anti-NF-kB p65 antibody (ab16502) at 0.5 µg/ml

    Lane 1 : Hela whole cell lysate
    Lane 2 : A431 whole cell lysate
    Lane 3 : Hela whole cell lysate with Human NF-kB p65 peptide (ab16636) at 1 µg/ml
    Lane 4 : A431 whole cell lysate with Human NF-kB p65 peptide (ab16636) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/5000 dilution

    Performed under reducing conditions.

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

  • 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

文献

This product has been referenced in:

  • Wang L  et al. Andrographolide impairs alpha-naphthylisothiocyanate-induced cholestatic liver injury in vivo. J Nat Med 73:388-396 (2019). Read more (PubMed: 30617707) »
  • Wixey JA  et al. Neuropathology in intrauterine growth restricted newborn piglets is associated with glial activation and proinflammatory status in the brain. J Neuroinflammation 16:5 (2019). Read more (PubMed: 30621715) »
See all 323 Publications for this product

客户评价及客户问答

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1-10 of 35 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (Human tissue)
Gel Running Conditions
Reduced Denaturing (10% Acrylamide gel)
Loading amount
20 µg
Treatment
UV B 100mJ/cm2
Specification
Human tissue
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

提交于 Apr 25 2019

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Prostate)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate
Permeabilization
No
Specification
Prostate
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

提交于 Jan 18 2019

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
HEK293
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

提交于 Dec 24 2018

Application
Western blot
Sample
Rat Cell lysate - whole cell (Heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Heart
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

提交于 Dec 24 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Cardiomyocytes)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Cardiomyocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

提交于 Dec 24 2018

Application
ChIP
Sample
Human Cell lysate - nuclear (Tumor Cell lines)
Negative control
GAPDH
Specification
Tumor Cell lines
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 30 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Positive control
IFNB1

Giorgos Sianidis

Verified customer

提交于 Dec 07 2018

Abreviews
Application
ChIP
Sample
Human Cell lysate - nuclear (Kidney (Clear Cell Renal Carcinoma))
Negative control
786-O clear cell renal carcinoma cells.
Specification
Kidney (Clear Cell Renal Carcinoma)
Detection step
Other
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
Metastatic renal cancer cell obtained from 786-O parental line.

Abcam user community

Verified customer

提交于 Aug 17 2018

Application
Western blot
Sample
Mouse Cell lysate - whole cell (BV-2 cells)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (8%)
Loading amount
40 µg
Treatment
1 μg/mL LPS for 24h
Specification
BV-2 cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Ms. Ji Hye Han

Verified customer

提交于 Jul 31 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Dog Cell (Immortalized histiocytic sarcoma (DH82))
Permeabilization
Yes - 0.1% Triton X-100 in PBS
Specification
Immortalized histiocytic sarcoma (DH82)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.1% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

提交于 Apr 16 2018

Application
Immunocytochemistry
Sample
Rat Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

提交于 Feb 27 2018

1-10 of 35 Abreviews

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