Anti-NF-kB p65抗体
参阅全部 NF-kB p65 一抗
兔多克隆抗体to NF-kB p65
Rabbit
适用于: ICC/IF, IHC-P, WB, IPmore details
与反应: Mouse, Human
预测可用于: Rat, Indian muntjac, Heterocephalus glaber
Synthetic peptide corresponding to Human NF-kB p65 aa 500 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
(Peptide available as ab16636)
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
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Liquid
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: 98.98% PBS, 1% BSA
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
浓度
批次浓度范围 100 µg 浓度为 0.9 - 1.03 mg/ml
Protein A purified
多克隆
IgG
Abpromise™承诺保证使用ab16502于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。
| 应用 | Ab评论 | 说明 |
|---|---|---|
| ICC/IF | (9) | Use a concentration of 1 - 5 µg/ml. |
| IHC-P | (4) | Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
| WB | (14) | Use a concentration of 0.5 µg/ml. Detects a band of approximately 64 kDa (predicted molecular weight: 60 kDa). Abcam recommends using milk as the blocking agent. |
| IP | (1) | Use at an assay dependent concentration. |
Entrez Gene: 5970 Human
Entrez Gene: 19697 Mouse
Omim: 164014 Human
SwissProt: Q04206 Human
SwissProt: Q04207 Mouse
Unigene: 502875 Human
Unigene: 249966 Mouse
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Western blot - Anti-NF-kB p65 antibody (ab16502)
All lanes : Anti-NF-kB p65 antibody (ab16502) at 1/1000 dilution
Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : NF-kB p65 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : A431 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 60 kDa
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.
Western blot - Anti-NF-kB p65 antibody (ab16502)
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 kDawhy 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)
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 BONDTM 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.
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-iTTM 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).
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 70oC; 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) - Anti-NF-kB p65 antibody (ab16502)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/
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of VillinCre;Dclk1f/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) - Anti-NF-kB p65 antibody (ab16502)Image from Qu D et al. PLoS ONE. 2015; 10(8): e0134212. Fig 5. doi:10.1371/journal.pone.0134212.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Dclk1f/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.
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.
Western blot - Anti-NF-kB p65 antibody (ab16502)
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 kDawhy is the actual band size different from the predicted?
Exposure time: 8 minutes
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody (ab16502)This image is courtesy of an anonymous abreview.
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.
Western blot - Anti-NF-kB p65 antibody (ab16502)
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 kDawhy 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
Western blot - Anti-NF-kB p65 antibody (ab16502)
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 kDawhy is the actual band size different from the predicted?
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.
