NF-KB p65 抗体 (YA267)

NF-KB p65 Antibody (YA267) is a Rabbit-derived and non-conjugated IgG monoclonal antibody, targeting to NF-KB p65.

NFkB p65（RELA/p65）是 NF-kappa-B 转录因子家族的核心成员之一，通常与 NFKB1/p50 形成异源二聚体 RELA-NFKB1，作为最丰富的 NF-kappa-B 复合物形式。该转录因子通过结合靶基因 DNA 上的 kappa-B 位点调控炎症、免疫、细胞分化、肿瘤发生等多种生物过程。在静息状态下，NF-kappa-B 与 I-kappa-B 抑制剂家族结合滞留于胞质；当 IKK 磷酸化降解 I-kappa-B 后，活化的 NF-kappa-B 转位入核发挥转录激活功能。RELA 的弱 DNA 结合位点可辅助复合物直接结合 DNA，同时通过改变染色质可及性间接调控基因表达。它还与 DDX1 协同结合 NF-kappa-B 启动子区，对 T 细胞中细胞因子基因表达至关重要（by similar）。此外，RELA-RELA 同源二聚体参与 invasin 介导的 IL-8 表达激活，并在 SARS-CoV-2 感染期间调控 IFN 应答中起关键作用。

Free

Q04206

Predicted band size: 65 kDa; Observed band size: 65 kDa

Protein A affinity purified.

Nucleus, Cytoplasm.

Non-conjugated

Unmodified

AB_3102376

Cell Biology

Primary Antibody; Recombinant Rabbit Monoclonal Antibody

Recombinant, Monoclonal

Rabbit

Human, Mouse

WB: 1:500-1:2000; ICC/IF: 1:50-1:200; IHC-P: 1:50-1:200; FC: 1:50-1:100; IP: Use at an assay dependent concentration.

• 
  Western blot analysis of extracts from Hela (lane 2(20μg) , A549 (lane 3(20μg) , RAW264.7 (lane 4(20μg),using NF-KB p65 Antibody (HY-P80245). Proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBST for 2 hour at room temperature. The primary antibody and Loading control antibody (Beta Actin, HY-P80438, 1/3000) was used in 5% BSA in TBST at 4°C overnight. Goat Anti-Mouse/Rabbit IgG-HRP Secondary Antibody (HY-P8004/HY-P8001, 1/10,000) was used for 1 hour at room temperature.
• 
  Immunocytochemistry analysis of HepG2 cells labeling NF-KB p65 with NF-KB p65 Antibody (HY-P80245) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with NF-KB p65 Antibody (HY-P0245) at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4 ℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002, Green) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. The Nuclear counterstain was DAPI (Blue).
• 
  Immunocytochemistry analysis of A549 cells labeling NF-KB p65 with NF-KB p65 Antibody (HY-P80245) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with NF-KB p65 Antibody (HY-P80245)at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4 ℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002,Green) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. The Nuclear counterstain was DAPI (Blue).
• 
  Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue using NF-KB p65 Antibody (YA267). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80245, 1/200) in 4℃ overnight. 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.
• 
  Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue using NF-KB p65 Antibody (YA267). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80245, 1/200) in 4℃ overnight. 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.
• 
  Flow cytometric analysis of 1X10^6 Hela cells labeling NF-KB p65 Antibody(HY-P80245, red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Then stained with the primary antibody at 1/1000 dilution for an hour at 4℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8002) was used as the secondary antibody at 1/1,000 dilution for 30 minutes at 4℃. Rabbit IgG Isotype Control (HY-P80879, blue) was used as the isotype control, cells without incubation with primary antibody were used as the unlabeled control (black).
• 
  Immunohistochemical analysis of paraffin-embedded human Pancreatic carcinoma‌ tissue using NF-KB p65 antibody was performed. The section was pretreated using high-temperature and high-pressure mediated EDTA antigen retrieval buffer (pH 9.0), for 5 minutes. The tissues were incubated with primary antibody (HY-P80245, 1:100 dilution) at room temperature for 60 minutes. Detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. The tissues were counterstained with hematoxylin and mounted with neutral balsam mounting medium.
• 
  Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue using NF-KB p65 antibody was performed. The section was pretreated using high-temperature and high-pressure mediated EDTA antigen retrieval buffer (pH 9.0), for 5 minutes. The tissues were incubated with primary antibody (HY-P80245, 1:100 dilution) at room temperature for 60 minutes. Detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. The tissues were counterstained with hematoxylin and mounted with neutral balsam mounting medium.
• 
  Tyramide signaling amplification based immunofluorescence was performed on paraffin-embedded human ovarian carcinoma (sample 1) tissue using NF-KB p65 antibody. Antigen retrieval was performed in EDTA buffer pH 9.0 (95 °C, 20 min) followed by cooling to RT. Then incubated with primary antibody (HY-P80245, 1:100 dilution) at room temperature for 60 minutes and HRP conjugated secondary antibody for 10 minutes. Fluorescence was then developed with TSA520 . The tissues were counterstained with DAPI and mounted with Anti-fade mounting medium.
• 
  Tyramide signaling amplification based immunofluorescence was performed on paraffin-embedded human ovarian carcinoma (sample 2) tissue using NF-KB p65 antibody. Antigen retrieval was performed in EDTA buffer pH 9.0 (95 °C, 20 min) followed by cooling to RT. Then incubated with primary antibody (HY-P80245, 1:100 dilution) at room temperature for 60 minutes and HRP conjugated secondary antibody for 10 minutes. Fluorescence was then developed with TSA520 . The tissues were counterstained with DAPI and mounted with Anti-fade mounting medium.

WB, ICC/IF, IHC-P, IP, FC

液体

Supplied in 1*TBS (pH7.4), 0.05% BSA and 40% Glycerol. Preservative: 0.05% Sodium Azide.

Stored at -20°C for 1 year. Avoid repeated freeze / thaw cycles.

Shipping with blue ice.

IgG

Endogenous

Synthetic peptide corresponding to Human NF-kB p65.AA range:490-540.