• 概述

• 产品描述 Proteins encoded by the v-Rel viral oncogene and its cellular homolog, c-Rel, are members of a family of transcription factors that include the two subunits of the transcription factor NF kB (p50 and p65) and the Drosophila maternal morphogen, dorsal. Both proteins specifically bind to DNA sequences that are the same or slight variations of the 10 bp kB sequence in the immunoglobulin k light chain enhancer. This same sequence is also present in a number of other cellular and viral enhancers. The DNA binding activity of NFkB is activated and NFkB is subsequently transported from the cytoplasm to the nucleus in cells exposed to mitogens or growth factors. cDNAs encoding precursors for two distinct proteins of the same size have been described, designated p105 and p100. The p105 precursor contains p50 at its N-terminus and a C-terminal region that when expressed as a separate molecule, designated pdI, binds to p50 and regulates its activity.
• 产品名称 Anti-NF-κB p65 Recombinant Rabbit Monoclonal Antibody [SZ10-04]
• 分子量 Predicted band size: 65 kDa
• 种属反应性 Human,Mouse
• 验证应用 WB,IF-Cell,IF-Tissue,IHC-P,IP,FC
• 抗体类型 重组兔单抗
• 免疫原 Synthetic peptide within human NF-kB p65 aa 490-540.
• 偶联 Non-conjugated

• 性能

• 形态 Liquid
• 浓度 1 mg/mL.
• 存放说明 Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
• 存储缓冲液 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
• 亚型 IgG
• 纯化方式 Protein A affinity purified.
• 亚细胞定位 Nucleus, Cytoplasm.
• 数据链接 SwissProt: Q04206 Human
  SwissProt: Q04207 Mouse
  
• 其它名称
  • 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
  • NF-κBp65
  more

• 应用

  WB: 1:1,000
  IF-Cell: 1:50-1:200
  IF-Tissue: 1:50-1:200
  IHC-P: 1:50-1:200
  FC: 1:50-1:100
  IP: Use at an assay dependent concentration.

• 

  Fig1: All lanes: Western blot analysis of NF-κB p65 with anti-NF-κB p65 antibody [SZ10-04] (ET1603-12) at 1:500 dilution.
  Lane 1: Wild-type Hela whole cell lysate (20 µg).
  Lane 2: NF-κB p65 knockout Hela whole cell lysate (20 µg).
  
  ET1603-12 was shown to specifically react with NF-κB p65 in wild-type Hela cells. No band was observed when NF-κB p65 knockout sample was tested. Wild-type and NF-κB p65 knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1603-12, 1/500) and Loading control antibody (Rabbit anti-β-actin, R1207-1, 1/1,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

  

  Fig2: Western blot analysis of NF-κB p65 on different lysates with Rabbit anti-NF-κB p65 antibody (ET1603-12) at 1/1,000 dilution.
  
  Lane 1: A549 cell lysate
  Lane 2: MCF7 cell lysate
  Lane 3: HeLa cell lysate
  Lane 4: RAW264.7 cell lysate
  
  Lysates/proteins at 15 µg/Lane.
  
  Predicted band size: 65 kDa
  Observed band size: 65 kDa
  
  Exposure time: 49 seconds;
  
  4-20% SDS-PAGE gel.
  
  Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1603-12) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

  

  Fig3: Immunocytochemistry analysis of HeLa cells treated with or without 50 ng/mL TNF-alpha for 20 minutes labeling NF-κB p65 with Rabbit anti-NF-κB p65 antibody (ET1603-12) at 1/100 dilution.
  
  Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-NF-κB p65 antibody (ET1603-12) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.
  
  Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  

  Fig4: ICC staining of NF-κB p65 in A549 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1603-12, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  

  Fig5: ICC staining of NF-κB p65 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1603-12, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  

  Fig6: Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma tissue with Rabbit anti-NF-κB p65 antibody (ET1603-12) at 1/400 dilution.
  
  The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-12) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  

  Fig7: Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-NF-κB p65 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-12, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  

  Fig8: Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-NF-κB p65 antibody (ET1603-12) at 1/400 dilution.
  
  The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1603-12) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  

  Fig9: Flow cytometric analysis of NF-κB p65 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1603-12, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).

  

  Fig10: NF-κB p65 was immunoprecipitated from 0.5 mg Hela whole cell lysates with ET1603-12 at 2 μg/mL. Western blot was performed from the immunoprecipitate using ET1603-12 at 1/500 dilution for 45 minutes at room temperature. Goat anti-Rabbit IgG-HRP Secondary Antibody (HA1001) was used at 1:300,000 dilution for 30 minutes at room temperature.
  
  Lane 1: Hela whole cell lysates at 10 μg;
  Lane 2: NF-κB p65 (ET1603-12) IP in Hela whole cell lysates;
  Lane 3: Rabbit IgG instead of NF-κB p65 (ET1603-12) in Hela whole cell lysates.
  
  Predicted band size: 60 kDa
  Observed band size: 65 kDa
  
  Exposure time: 10 seconds;
  
  8% SDS-PAGE gel.

• 背景文献

• 1. Kang K et al. Carnosic acid slows photoreceptor degeneration in the Pde6b(rd10) mouse model of retinitis pigmentosa. Sci Rep 6:22632 (2016).
  
  2. Kropp KA et al. A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection. PLoS Pathog 11:e1004737 (2015).

• Publishing research using Anti-NF-κB p65 Recombinant Rabbit Monoclonal Antibody? Please let us know so that we can cite the reference in this datasheet.

• PMID:
  37024790 Experimental study on the effect of luteolin on the proliferation, apoptosis and expression of inflammation-related mediators in lipopolysaccharide-induced keratinocytes Author: Wang, X., Yao, Y., Li, Y., Guo, S., Li, Y., & Zhang, G. Published: 2023 Application: WB Species: Human Journal: Int J Immunopathol Pharmacol IF: 3.298
• PMID:
  35115492 TNF antagonist sensitizes synovial fibroblasts to ferroptotic cell death in collagen-induced arthritis mouse models Author: Wu, J., Feng, Z., Chen, L., Li, Y., Bian, H., Geng, J., Zheng, Z. H., Fu, X., Pei, Z., Qin, Y., Yang, L., Zhao, Y., Wang, K., Chen, R., He, Q., Nan, G., Jiang, X., Chen, Z. N., & Zhu, P. Published: 2022 Application: WB Species: Mouse Journal: Nat Commun IF: 14.919
• PMID:
  35266845 TRAF6 autophagic degradation by avibirnavirus VP3 inhibits antiviral innate immunity via blocking NFKB/NF-κB activation Author: Deng, T., Hu, B., Wang, X., Ding, S., Lin, L., Yan, Y., Peng, X., Zheng, X., Liao, M., Jin, Y., Dong, W., Gu, J., & Zhou, J. Published: 2022 Application: WB Species: Chicken Journal: Autophagy IF: 16.016
• PMID:
  35651980 BRAFV600E Mutation-Responsive miRNA-222-3p Promotes Metastasis of Papillary Thyroid Cancer Cells via Snail-Induced EMT Author: Gao, Y., Xiang, D., Li, W., Zheng, X., Wang, L., Li, Z., & Chen, T. Published: 2022 Application: WB Journal: Front Endocrinol (Lausanne). IF: 5.555
• PMID:
  35538475 N6-methyladenosine-modified TRAF1 promotes sunitinib resistance by regulating apoptosis and angiogenesis in a METTL14-dependent manner in renal cell carcinoma Author: Chen, Y., Lu, Z., Qi, C., Yu, C., Li, Y., Huan, W., Wang, R., Luo, W., Shen, D., Ding, L., Ren, L., Xie, H., Xue, D., Wang, M., Ni, K., Xia, L., Qian, J., & Li, G. Published: 2022 Application: WB Species: Human Journal: Mol Cancer. IF: 41.444
• PMID:
  33716743 Ginsenoside Rb1 Alleviates Alcohol-Induced Liver Injury by Inhibiting Steatosis, Oxidative Stress, and Inflammation. Frontiers in pharmacology, 12, 616409. Author: Lai, Y., Tan, Q., Xv, S., Huang, S., Wang, Y., Li, Y., Zeng, T., Mo, C., Chen, Y., Huang, S., Zhou, C., Gao, L., & Lv, Z. Published: 2021 Application: IHC,IF Species: Zebrafish Journal: Front Pharmacol IF: 5.81
• PMID:
  34051663 Autophagy was activated against the damages of placentas caused by nano-copper oral exposure. Ecotoxicology and environmental safety, 220, 112364. Advance online publication. Author: Kang, M., Luo, J., Zhao, L., Shi, F., Ye, G., He, X., Hao, S., Yang, D., Chen, H., Guo, H., Li, Y., & Tang, H. Published: 2021 Application: WB Species: Rat Journal: Ecotoxicology and Environmental Safety IF: 4.872
• PMID:
  34707127 Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling Author: Wang, J., Wang, J., Hong, W., Zhang, L., Song, L., Shi, Q., Shao, Y., Hao, G., Fang, C., Qiu, Y., Yang, L., Yang, Z., Wang, J., Cao, J., Yang, B., He, Q., & Weng, Q. Published: 2021 Application: WB Species: Mouse Journal: Nat Commun IF: 14.914
• PMID:
  30818128 Fusobacterium nucleatum stimulates monocyte adhesion to and transmigration through endothelial cells Author: Yafei Wu Published: 2019 Application: WB Species: HUVEC cell Journal: Arch Oral Biol. IF: 1.663
• PMID:
  30396897 Phosphothreonine Lyase Promotes p65 Degradation in a Mitogen-Activated Protein Kinase/Mitogen- and StressActivated Protein Kinase 1-Dependent Manner Author: Qin Liu Published: 2019 Application: WB Species: Escherichia coli Journal: Infection and Immunity IF: 3.256
• PMID:
  30448447 Astaxanthin protects lipopolysaccharide-induced inflammatory response in Channa argus through inhibiting NF-κB and MAPKs signaling pathways. Author: wangguiqing Published: 2018 Application: WB Species: Channa argus Journal: Fish Shellfish Immunol. IF: 3.185

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Specific Protocols

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.