NF-KB p65 Antibody (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) is a core component of the NF-kappa-B transcription factor family, predominantly forming the heterodimeric RELA-NFKB1 complex with NFKB1/p50 as the most abundant NF-kappa-B variant. This pleiotropic factor binds to kappa-B sites in target gene DNA to regulate diverse biological processes including inflammation, immunity, cell differentiation, and tumorigenesis. Inactive cytoplasmic NF-kappa-B complexes are retained by I-kappa-B inhibitors until IKK-mediated phosphorylation triggers their nuclear translocation for transcriptional activation. RELA contributes weak DNA-binding activity to the complex while also modulating chromatin accessibility for indirect gene regulation. It cooperates with DDX1 at NF-kappa-B promoter regions and is essential for cytokine expression in T-cells (by similar). The RELA-RELA homodimer mediates invasin-induced IL-8 expression and serves as a key regulator of IFN response during SARS-CoV-2 infection.

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: 1:500-1:2,000; ICC/IF: 1:50-1:200; IHC-P: 1:50-1:200; FC: 1:50-1:100; IP: Use at an assay dependent concentration.

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  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

Liquid

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.