重组Anti-NF-kB p65抗体[E379] (ab32536)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E379] to NF-kB p65
- Suitable for: WB, IHC-P, ICC/IF, IP
- Knockout validated
- Reacts with: Mouse, Human
概述
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产品名称
Anti-NF-kB p65抗体[E379]
参阅全部 NF-kB p65 一抗 -
描述
兔单克隆抗体[E379] to NF-kB p65 -
宿主
Rabbit -
特异性
ab32536 recognises NF-kB p65. The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse. This antibody is unsuitable for detecting NF-KB p65 in mouse tissue lysates. The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230. Mouse brain and spleen are positive reported by PMID: 21479220 and 20008488 -
Tested Applications & Species
Application Species ICC/IF HumanIHC-P HumanIP HumanWB MouseHuman -
免疫原
Synthetic peptide within Human NF-kB p65 aa 500 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: Q04206 -
阳性对照
- WB: Wild-type HAP1 cell lysate. HeLa, MEF, RAW 264.7 and A431 cell lysate; Human fetal brain, kidney and lung tissue lysates. IHC-P: Human breast carcinoma and colon carcinoma tissue. Mouse colon tissue. ICC/IF: HeLa cells. IP: NF-kB p65 IP in HeLa whole cell lysate (ab150035).
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常规说明
Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).
See other anti-rabbit secondary antibodies that can be used with this antibody.
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
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.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
E379 -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
- Alexa Fluor® 488 Anti-NF-kB p65 antibody [E379] (ab190205)
- Alexa Fluor® 647 Anti-NF-kB p65 antibody [E379] (ab190589)
- Biotin Anti-NF-kB p65 antibody [E379] (ab205823)
- Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)
- PE Anti-NF-kB p65 antibody [E379] (ab208750)
- Alexa Fluor® 555 Anti-NF-kB p65 antibody [E379] (ab214634)
- Alexa Fluor® 594 Anti-NF-kB p65 antibody [E379] (ab214846)
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Assay kits
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
应用
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32536 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
应用 | Species |
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ICC/IF |
Human
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IHC-P |
Human
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IP |
Human
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WB |
Mouse
Human
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应用 | Ab评论 | 说明 |
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WB | (11) |
1/1000 - 1/100000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).
This antibody might not detect target band in mouse tissues. |
IHC-P |
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse. |
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ICC/IF | (2) |
1/100.
For unpurified use at 1/250 -1/500. |
IP |
1/30.
For unpurified use at 1/200. |
说明 |
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WB
1/1000 - 1/100000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa). This antibody might not detect target band in mouse tissues. |
IHC-P
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse. |
ICC/IF
1/100. For unpurified use at 1/250 -1/500. |
IP
1/30. For unpurified use at 1/200. |
靶标
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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
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数据库链接
- Entrez Gene: 5970 Human
- Entrez Gene: 19697 Mouse
- Omim: 164014 Human
- SwissProt: Q04206 Human
- SwissProt: Q04207 Mouse
- Unigene: 502875 Human
- Unigene: 249966 Mouse
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别名
- Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
- MGC131774 antibody
- NF kappa B p65delta3 antibody
see all
图片
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Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NF-kB 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 - ab32536 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.
Unpurified ab32536 was shown to specifically react with NF-kB p65 in wild-type HAP1 cells. No band was observed when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 (NF-kB p65) and ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively 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 h at room temperature before imaging. -
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab32536 at 1:100 dilution.
Cells were fixed in 100% methanol. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain.
PBS instead of the primary antibody was used as the secondary antibody only control.
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All lanes : Anti-NF-kB p65 antibody [E379] (ab32536) 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: 65 kDa
Observed band size: 65 kDaBlocking/Diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 37 second for Lane1 and 3 seconds for Lanes 2 and 3
Normal brain might express low level of p65 (PMID: 21479220)
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)Souslova et al PLoS One. 2010 Mar 25;5(3):e9902. doi: 10.1371/journal.pone.0009902. Fig 2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
MMP3 and NFκB are co-localized in human atherosclerotic plaques.
Sections (4 µm) of paraffin embedded tissue blocks of atherosclerotic plaques were subjected to double immunostaining of MMP3 and p50 (A, B, and C) or MMP3 and p65 (D, E and F, shown), using rabbit polyclonal NFκB p50 and MMP3 antibodies (Abcam) and ab32536, respectively, and goat anti-rabbit secondary antibodies.
The double immunostaining was visualized with Liquid Permanent Red chromogen and Vector Blue chromogen. Pink colour indicates expression of p50 (A, B and C) or p65 (D, E and F). Blue colour indicates expression of MMP3 (A, B, C, D, E and F.
Arrow indicates cell co-expressing MMP3 with p50 (B and C) or with p65 (E and F).
200× magnification in A and D; 400× magnification in B, C, E and F.
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Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)Fazalul Rahiman et al PLoS One. 2016 Apr 7;11(4):e0153005. doi: 10.1371/journal.pone.0153005. eCollection 2016. Fig 1. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
NF-κB/p65 nuclear localisation in differentiated THP-1 (Human monocytic leukemia cell line) cells.
Cells were fixed, permeabilized, stained with ab32536 and visualised with Alexa Fluor® 555 goat anti-rabbit IgG (green). The nuclei were counterstained with DAPI (blue). Microscopy images A. Inactive NF-κB/p65 proteins localization in the cytoplasm of the non-stimulated THP-1 cells (top row) and B. Translocated NF-κB/p65 proteins into the nuclei of THP-1 cells following LPS stimulation (bottom row).
Images were acquired for each fluorescence channel, using suitable TRITC and DAPI filters and a 63× objective.
Scale bar: 10μm
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)Image from Bel S et al., J Biol Chem. 2012 Jul 20;287(30):25631-9. Epub 2012 May 2. Fig 8.; doi: 10.1074/jbc.M112.364786; July 20, 2012, The Journal of Biological Chemistry, 287, 25631-25639.
Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with unpurified ab32536.
Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit. -
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)Image from Ali, Ahmed Atef Ahmed et al. PLoS ONE 11.4 (2016): e0154278. Fig #8. doi: 10.1371/journal.pone.0154278. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536 (Middle panel).
Briefly, the tested cells were seeded on coverslips treated with HCl and ethanol, and autoclaved prior to use. Immunostaining of the p65 subunit of NF-κB was done by permeabilizing the cells with Triton X-10, then by treating the cells with anti-NF-κB p65 rabbit monoclonal primary antibody [E379] (ab32536), followed by Alexa Fluor® 488 Donkey anti-rabbit IgG secondary antibody. Nuclei of cells were stained with DAPI (Left panel).
Merge (Right panel).
Images were acquired using fluorescence microscope.
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Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells using unpurified ab32536.
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All lanes : Anti-NF-kB p65 antibody [E379] (ab32536) at 1000 µg
Lane 1 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 2 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharides for 6 hours whole cell lysates
Lane 3 : Mouse brain lysates
Lane 4 : Mouse spleen lysates
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 65 kDa
Exposure time: 3 secondsThis antibody is unsuitable for detecting mouse tissue lysates.
The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230.
Mouse brain and spleen are positive reported by PMID: 21479220 and 20008488
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Anti-NF-kB p65 [E379] antibody (unpurified ab32536) reactivity with reduced wild type (WT) and p65 knockout (KO) mouse embryonic fibroblast (MEF) lysate.
After SDS-PAGE, membranes were blocked in 5% milk in TBS + 0.1% Tween for 1 hour at 25°C before incubation with unpurified ab32536 (1:1,000 dilution in 5% milk TBS + 0.1% Tween) for 16 hours at 4ºC. Blots was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue sections labeling NF-kB p65 with Purified ab32536 at 1:2000 dilution (0.2 μg/ml).
Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used.
PBS instead of the primary antibody was used as the negative control.
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ab32536 (purified) at 1:30 dilution (2 μg) immunoprecipitating NF-kB p65 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa whole cell lysate 10μg
Lane 2 (+): ab32536 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32536 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST. -
Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 65 kDaBlocking and diluting buffer: 5% NFDM/TBST.
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Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution (purified) + MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysates at 15 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 65 kDaBlocking and diluting buffer: 5% NFDM/TBST.
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Anti-NF-kB p65 antibody [E379] (ab32536) at 1/100000 dilution (unpurified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Predicted band size: 65 kDa
Observed band size: 65 kDa -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab32536.
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
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SDS download
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Datasheet download
文献 (146)
ab32536 被引用在 146 文献中.
- Zhu H et al. CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-a in TMJ osteoarthritis. Cell Death Dis 11:284 (2020). PubMed: 32332704
- Li Q et al. Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-?B (p65) DNA binding and promoted periodontal repair in rats. J Cell Physiol N/A:N/A (2020). PubMed: 32399963
- Li L et al. MicroRNA-584 Impairs Cellular Proliferation and Sensitizes Osteosarcoma Cells to Cisplatin and Taxanes by Targeting CCN2. Cancer Manag Res 12:2577-2587 (2020). PubMed: 32346311
- Kong Q et al. Autophagy inhibits TLR4-mediated invasiveness of oral cancer cells via the NF-?B pathway. Oral Dis N/A:N/A (2020). PubMed: 32291890
- Maisto R et al. Resolvin D1 Modulates the Intracellular VEGF-Related miRNAs of Retinal Photoreceptors Challenged With High Glucose. Front Pharmacol 11:235 (2020). PubMed: 32210819