Recombinant Anti-NF-kB p65 antibody [E379] (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.

Unpurified 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.

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

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 37 second for Lane1 and 3 seconds for Lanes 2 and 3

Normal brain might express low level of p65 (PMID: 21479220)

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).

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

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

MMP3 and NFκB are co-localized in human atherosclerotic plaques.

Sections (4 µm) of paraffin embedded tissue blocks of atherosclerotic plaques were subjected to double immunostaining of MMP3 and p50 (A, B, and C) or MMP3 and p65 (D, E and F, shown), using rabbit polyclonal NFκB p50 and MMP3 antibodies (Abcam) and ab32536, respectively, and goat anti-rabbit secondary antibodies.

The double immunostaining was visualized with Liquid Permanent Red chromogen and Vector Blue chromogen. Pink colour indicates expression of p50 (A, B and C) or p65 (D, E and F). Blue colour indicates expression of MMP3 (A, B, C, D, E and F.

Arrow indicates cell co-expressing MMP3 with p50 (B and C) or with p65 (E and F).

200× magnification in A and D; 400× magnification in B, C, E and F.

NF-κB/p65 nuclear localisation in differentiated THP-1 (Human monocytic leukemia cell line) cells.

Cells were fixed, permeabilized, stained with ab32536 and visualised with Alexa Fluor^® 555 goat anti-rabbit IgG (green). The nuclei were counterstained with DAPI (blue). Microscopy images A. Inactive NF-κB/p65 proteins localization in the cytoplasm of the non-stimulated THP-1 cells (top row) and B. Translocated NF-κB/p65 proteins into the nuclei of THP-1 cells following LPS stimulation (bottom row).

Images were acquired for each fluorescence channel, using suitable TRITC and DAPI filters and a 63× objective.

Scale bar: 10μm

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

Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536 (Middle panel).

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 (Left panel).

Merge (Right panel).

Images were acquired using fluorescence microscope. 

Immunofluorescent staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells using unpurified ab32536.

This antibody is unsuitable for detecting mouse tissue lysates.

The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230.

Although some papers support the expression of NF-κB p65 in mouse tissue (PMID: 21479220 and 20008488), ab32536 cannot detect band of interest in these mouse tissue.

Anti-NF-kB p65 [E379] antibody (unpurified ab32536) reactivity with reduced wild type (WT) and p65 knockout (KO) mouse embryonic fibroblast (MEF) lysate.

After SDS-PAGE, membranes were blocked in 5% milk in TBS + 0.1% Tween for 1 hour at 25°C before incubation with unpurified ab32536 (1:1,000 dilution in 5% milk TBS + 0.1% Tween) for 16 hours at 4ºC. Blots was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.

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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.

PBS instead of the primary antibody was used as the negative control.

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

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

Blocking and diluting buffer: 5% NFDM/TBST.

Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab32536.