NF-kB p65 Recombinant Rabbit Monoclonal Antibody [PSH0-27]

Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/2,000 dilution.

Lane 1: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 2: A431 (Human epidermoid carcinoma skin squamous cell) cell lysate
Lane 3: Raji (Human Burkitt's lymphoma cell) cell lysate
Lane 4: NIH/3T3 (Mouse fibroblast) cell lysate
Lane 5: C2C12 (Mouse myoblast) cell lysate
Lane 6: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate

Lysates/proteins at 20 µg/Lane.
Exposure time: 25 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA721307, 1/2,000 in 5% NFDM/TBST, overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

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

☑ Knockout (KO)

Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/500 dilution.

Lane 1: HeLa cell lysate (control) (35 µg/Lane)
Lane 2: HeLa-RELA-KO (1) (35 µg/Lane)
Lane 3: HeLa-RELA-KO (2) (35 µg/Lane)

Predicted band size: 60 kDa
Observed band size: 65 kDa

Exposure time: 3 minutes;
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 (HA721307) at 1/500 dilution was used in 5% NFDM/TBST 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.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/1,000 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 (HA721307) at 1/1,000 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 breast tissue with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/1,000 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 (HA721307) at 1/1,000 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.

Immunocytochemistry analysis of HeLa cells labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of NIH/3T3 cells treated with or without 50μg/mL TNF-α for 20 minutes labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 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 (HA721307) at 1/200 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. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

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

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1/1,000) (red) 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).

Flow cytometric analysis of NIH/3T3 cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1ug/ml) (red) 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).

Flow cytometric analysis of PC-12 cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1/1,000) (red) 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).