Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NFκB 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 - ab16502 observed at 70 kDa. Red - ab8245 loading control, observed at 37 kDa.

ab16502 was shown to react with NFκB p65 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when NFκB p65 knockout samples were used. Wild-type and NFκB p65 knockout samples were subjected to SDS-PAGE. ab16502 (NFκB p65) and ab8245 (loading control to GAPDH) were both diluted 1/1000 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 hour at room temperature before imaging.

All lanes : Anti-NF-kB p65 antibody (ab16502) at 1/1000 dilution

Lane 1 : Human fetal brain lysates
Lane 2 : Human fetal kidney lysates
Lane 3 : Human fetal lung lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 60 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

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

Exposure Time: 15 seconds for Lane1 and 3 seconds for Lanes 2 and 3

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

IHC image of Anti-NF-kB p65 antibody staining in a section of formalin-fixed paraffin-embedded human breast adenocarcinoma performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab16502, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

ICC/IF image of ab16502 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16502, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 phalloidin was used to label F-actin (red).

NF-kB p65 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to NFkB p65 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab16502.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody Anti-Rabbit HRP (IgG light chain) (ab99697).
Band: 68kDa: NFkB p65

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Villin^Cre;Dclk1^f/f mouse colon tissue sections labeling NF-kB p65 with ab16502 (brown). Alcian blue was used for counterstaining.Heat-induced epitope retrieval was performed on 4-μm formalin-fixed paraffin-embedded sections by utilizing a pressurized Decloaking Chamber in citrate buffer (pH 6.0) at 99°C for 18 min. For brightfield microscopy, slides were exposed to peroxidase blocking solution prior to the addition of primary antibody (ab16502). After incubation with primary antibody overnight at 4°C, the slides were incubated in peroxidase-conjugated polymer.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Dclk1^f/f mouse colon tissue sections labeling NF-kB p65 with ab16502 (brown). Alcian blue was used for counterstaining.Heat-induced epitope retrieval was performed on 4-μm formalin-fixed paraffin-embedded sections by utilizing a pressurized Decloaking Chamber in citrate buffer (pH 6.0) at 99°C for 18 min. For brightfield microscopy, slides were exposed to peroxidase blocking solution prior to the addition of primary antibody (ab16502). After incubation with primary antibody overnight at 4°C, the slides were incubated in peroxidase-conjugated polymer.

 

ICC/IF image of ab16502 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab16502, 1µg/ml) overnight at +4°C. The secondary antibody (green) was goat anti-rabbit DyLight® 488 (IgG - H&L, pre-adsorbed) (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1:200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-NF-kB p65 antibody (ab16502) at 1 µg/ml

Lane 1 : Spleen (Mouse) Tissue Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 64 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

ab16502 staining NF-kB p65 in murine peritoneal tumour cells by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 1% BSA for 60 minutes at room temperature. Samples were incubated with primary antibody (1/1000) for 2 hours. An undiluted Alexa Flour^®647-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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Anti-NF-kB p65 antibody (ab16502) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 50000 mg/ml

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 64 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab16502 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

All lanes : Anti-NF-kB p65 antibody (ab16502) at 0.5 µg/ml

Lane 1 : Hela whole cell lysate
Lane 2 : A431 whole cell lysate
Lane 3 : Hela whole cell lysate with Human NF-kB p65 peptide (ab16636) at 1 µg/ml
Lane 4 : A431 whole cell lysate with Human NF-kB p65 peptide (ab16636) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 64 kDa why is the actual band size different from the predicted?

ab16502 at a 1/500 dilution staining Asynchronous and paraformaldehyde-fixed (4%) HeLa cells by immunocytochemistry. The antibody was incubated with the cells 30 minutes and then detected using a Cy3 conjugated Goat Anti-Mouse IgG (H+L) antibody.

This image is courtesy of an Abreview by Kirk McManus submitted on 27 February 2006.

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