Recombinant Anti-NF-kB p65 antibody [E379] - BSA and Azide free (ab207297)

This WB data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NF-kB p65 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32536 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.

ab32536 was shown to specifically react with NF-kB p65 in wild-type HAP1 cells. No band was observed when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 (NF-kB p65) and ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab32536 at 1:100 dilution. Cells were fixed in 100% Methanol. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse spleen. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling NF-kB p65 with ab32536 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing the signal translocated from the cytoplasm into the nucleus in NIH/3T3 cells after the treatment with TNF-alpha (50 ng/ml) for 20 min. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution in treated (right) and untreated cells (left).

ab32536 (purified) at 1:30 dilution (2μg) immunoprecipitating NF-kB p65 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab32536 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32536 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human tonsil. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

This data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (ab32536).

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human prostatic hyperplasia. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon carcinoma tissue sections labeling NF-kB p65 with Purified ab32536 at 1:2000 dilution (0.2 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (Alexa Fluor® 647). Please refer to ab190589 for protocol details.

ab190589 staining NF-kB p65 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab190589 at a working dilution of 1 in 100 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal in 100% methanol (5 min) fixed HeLa cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (PE). Please refer to ab208750 for protocol details.

ab208750 staining NF-kB p65 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab208750 at 1/500 dilution (Pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 100% methanol (5min).

Clone E379 (ab207297) has been successfully conjugated by Abcam. This image was generated using Anti-NF-kB p65 antibody [E379] (Alexa Fluor® 488). Please refer to ab190205 for protocol details.

ab190205 staining NF-kB p65 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab190205 at a working dilution of 1 in 50 (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 594, shown in red) at a dilution of 1 in 250 overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal in 4% formaldehyde (10 min) fixed HeLa cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536. Briefly, the tested cells were seeded on coverslips treated with HCl and ethanol, and autoclaved prior to use. Immunostaining of the p65 subunit of NF-κB was done by permeabilizing the cells with Triton X-10, then by treating the cells with anti-NF-κB p65 rabbit monoclonal primary antibody [E379] (ab32536), followed by Alexa Fluor^® 488 Donkey anti-rabbit IgG secondary antibody. Nuclei of cells were stained with DAPI. Images were acquired using fluorescence microscope. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

Immunohistochemical analysis of paraffin-embedded human Breast carcinoma using unpurified anti-NF-kB p65 Rabbit Monoclonal Antibody

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

Immunofluorescent staining of HeLa cells using anti-NF-kB p65 Rabbit Monoclonal Antibody ( unpurified ab32536)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32536).

This IHC data was generated using the same anti-NF-kB p65 antibody clone, E379, in a different buffer formulation (cat# ab32536).

Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with ab32536.

Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit.