NF-kB p65 Recombinant Rabbit Monoclonal Antibody [SZ10-04] - BSA and Azide free

☑ Knockout (KO)

Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/5,000 dilution.

Lane 1: Wild-type HeLa cell lysate
Lane 2: HeLa KO cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 10 seconds; ECL: K1801;

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 (HA750058) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/5,000 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; ECL: K1801;

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 (HA750058) at 1/5,000 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.

☑ Knockdown (KD)

Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-NF-kB p65 KD cell lysate

Lysates/proteins at 10 µg/Lane.

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

Exposure time: 60 seconds; ECL: K1801;

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 (HA750058) at 1/5,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of HeLa cells treated with or without 50 ng/mL TNF-alpha for 20 minutes labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/400 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-kB p65 antibody (HA750058) at 1/400 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.

Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma tissue with Rabbit anti-NF-kB p65 antibody (HA750058) 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 (HA750058) 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.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA750058) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA750058) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA750058) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA750058) at 1/200 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.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Rabbit anti-NF-kB p65 antibody (HA750058) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA750058) at 1/200 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.

NF-κB p65 was immunoprecipitated from 0.5 mg Hela whole cell lysates with HA750058 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.

Flow cytometric analysis of HeLa cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750058, 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).