Anti-NF-kB p65 抗体

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody (AB16502)

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.

Image from Qu D et al. PLoS ONE. 2015; 10(8): e0134212. Fig 5. doi:10.1371/journal.pone.0134212. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• IHC

Lab

Immunohistochemistry - Anti-NF-kB p65 antibody (AB16502)

Immunohistochemical analysis of formalin fixed paraffin embedded human breast carcinoma labelling NF-kB p65 with ab16502 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC2) 100°C, pH 8.5 for 32 mins.

ab16502 Anti-NF-kB p65 antibody was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

AbReview4213****

Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody (AB16502)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody (AB16502)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody (AB16502)

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

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody (AB16502)

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).

• IP

Unknown

Immunoprecipitation - Anti-NF-kB p65 antibody (AB16502)

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

All lanes:

Immunoprecipitation - Anti-NF-kB p65 antibody (ab16502)

Predicted band size: 60 kDa

false

• IHC-P

AbReview52099****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody (AB16502)

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.

This image is courtesy of an anonymous abreview.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody (AB16502)

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.

Image from Qu D et al. PLoS ONE. 2015; 10(8): e0134212. Fig 5. doi:10.1371/journal.pone.0134212.

• WB

Lab

Western blot - Anti-NF-kB p65 antibody (AB16502)

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:

Western blot - Anti-NF-kB p65 antibody (ab16502) at 1 µg/mL

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 50000 mg/mL

Predicted band size: 60 kDa

Observed band size: 64 kDa

true

Exposure time: 4min

• WB

Unknown

Western blot - Anti-NF-kB p65 antibody (AB16502)

All lanes:

Western blot - Anti-NF-kB p65 antibody (ab16502) at 1 µg/mL

Lane 1:

Spleen (Mouse) Tissue Lysate at 10 µg

Lane 2:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 60 kDa

Observed band size: 64 kDa

true

Exposure time: 8min

• WB

Lab

Western blot - Anti-NF-kB p65 antibody (AB16502)

This blot was produced using a 4-12% Bis-tris gel under the MES 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 before being incubated with ab16502 overnight at 4°C. Antibody binding was detected using the Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) at a 1 : 10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

Merged signal (red and green). Green - ab16502 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa Goat anti-Mouse IgG H&L (IRDye^® 680RD) ab216776.

All lanes:

Western blot - Anti-NF-kB p65 antibody (ab16502) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

NF-kB p65 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

A431 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 36 kDa,60 kDa

false

• WB

Lab

Western blot - Anti-NF-kB p65 antibody (AB16502)

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)

All lanes:

Western blot - Anti-NF-kB p65 antibody (ab16502) at 1/1000 dilution

Lane 1:

Human fetal brain lysates at 20 µg

Lane 2:

Human fetal kidney lysates at 20 µg

Lane 3:

Human fetal lung lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 60 kDa

Observed band size: 65 kDa

false

• WB

Project

Western blot - Anti-NF-kB p65 antibody (AB16502)

All lanes:

Western blot - Anti-NF-kB p65 antibody (ab16502) at 0.5 µg/mL

Lanes 1 and 3:

Hela whole cell lysate at 20 µg

Lanes 2 and 4:

A431 whole cell lysate at 20 µg

Secondary

All lanes:

Alexa Fluor Goat polyclonal to Rabbit IgG at 1/5000 dilution

Predicted band size: 60 kDa

Observed band size: 64 kDa

false

• WB

CiteAb

Western blot - Anti-NF-kB p65 antibody (AB16502)

NF-kB p65 Western Blotting using Anti-NF-kB p65 antibody, ab16502. Publication image from Omer, A. et al., 2020, Nat Commun, PubMed : 33020468.

Depletion of G3BP1 impairs canonical SASP signaling through reduction of STAT3 and NF-κB signaling.WI-38 cells were treated with siRNA against G3BP1 (siG3BP1 #1 and #2) or scrambled control (siCTL) and assessed during proliferative stage (PRO) and 8-day post-ionizing radiation (+IR). a RNA was extracted and assayed by RT-qPCR using primers against indicated mRNAs. The data are a mean of three independent experiments ± s.e.m (two-tailed unpaired Student’s t test, exact < 0.001 p-values from left to right : 0.00095, 0.00022). b Conditioned media from PRO and from +IR WI-38 cells treated with siRNA against G3BP1 (siG3BP1) and scrambled control (siCTL) were analyzed by multiplex arrays. The heatmap indicates the fold change in comparison to the control PRO, SEN siCTL (siCTL), and SEN siG3BP1 (siG3BP1). Relative expression levels per replicate and average fold change differences are shown as indicated in heatmap. c (left) Cell lysates as described were subjected to western blot analysis against indicated proteins. (right) Quantifications represent a mean of relative protein levels from three independent experiments ± s.e.m (two-tailed unpaired Student’s t test, exact < 0.001 p-values from left to right : 0.0007). d WI-38 cells as described were analyzed by immunofluorescence with antibody against p65 during PRO and SEN. DAPI staining was used to visualize nuclei. Scale bar, 20 μm. Source Data for panels (a) and (c) are provided in the Source Data File. Raw data for (b) is provided in Supplementary Data 2.

false