antibody

Anti-NF-κB p65 Antibody [SZ10-04]

ET1603-12TR

20 uL

99 USD

monoclonal

domestic rabbit

nonconjugated

SZ10-04

human, mouse

western blot, immunoprecipitation, flow cytometry, immunohistochemistry - paraffin section

Anti-NF-κB p65 Antibody [SZ10-04]

Human,Mouse

WB,IF-Cell,IF-Tissue,IHC-P,IP,FC

Non-conjugated

Synthetic peptide within human NF-kB p65 aa 490-540.

SwissProt: Q04206 Human;SwissProt: Q04207 Mouse

Rabbit

SZ10-04

IgG

20 uL

99 USD

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

Liquid

Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.

Protein A affinity purified.

Recombinant Rabbit monoclonal Antibody

Hela whole cell lysate, A549 cell lysate, MCF7 cell lysate, HeLa cell lysate, RAW264.7 cell lysate, HeLa cells treated with 50 ng/mL TNF-alpha for 20 minutes, A549, NIH/3T3, human lung squamous cell carcinoma tissue, human breast carcinoma tissue,human lung tissue.

Predicted band size: 65 kDa

Nucleus, Cytoplasm.

1 mg/mL.

WB: 1:1,000-1:5,000 ;IF-Cell: 1:50-1:200 ;IF-Tissue: 1:50-1:200 ;IHC-P: 1:50-1:200 ;FC: 1:2,000 ;IP: Use at an assay dependent concentration.

ET1603-12_2.jpg

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.

ET1603-12_3.jpg

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.

ET1603-12_4.jpg

Flow cytometric analysis of HeLa cells labeling NF-κB p65. Cells were fixed and permeabilized. Then stained with the primary antibody (ET1603-12, red) at 1/2,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

ET1603-12_5.jpg

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).

ET1603-12_6.jpg

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).

ET1603-12_7.jpg

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.

ET1603-12_8.jpg

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.

ET1603-12_9.jpg

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.

ET1603-12_10.jpg

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.