Anti-NF-kB p65 抗体 (ab16502)
Key features and details
- Rabbit polyclonal to NF-kB p65
- Suitable for: ICC/IF, IHC-P, WB, IP
- Knockout validated
- Reacts with: Mouse, Human
- Isotype: IgG
リコンビナント抗体で、ロット間での高い再現性を実現
- 異なるロット間での安定した再現性
- 容易なスケールアップ
- 評価試験による特異性の確認済み
- 倫理基準に準拠 - アニマル・フリーの生産
製品の概要
-
製品名
Anti-NF-kB p65 antibody
NF-kB p65 一次抗体 製品一覧 -
製品の詳細
Rabbit polyclonal to NF-kB p65 -
由来種
Rabbit -
アプリケーション
適用あり: ICC/IF, IHC-P, WB, IPmore details -
種交差性
交差種: Mouse, Human
交差が予測される動物種: Rat, Indian muntjac, Heterocephalus glaber -
免疫原
Synthetic peptide corresponding to Human NF-kB p65 aa 500 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
(Peptide available asab16636) -
特記事項
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.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
製品の特性
-
製品の状態
Liquid -
保存方法
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
バッファー
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. -
Concentration information loading...
-
精製度
Protein A purified -
ポリ/モノ
ポリクローナル -
アイソタイプ
IgG -
研究分野
関連製品
-
Assay kits
-
Compatible Secondaries
-
Conjugation kits
-
Isotype control
-
Recombinant Protein
-
Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab16502の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
---|---|---|
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.
|
特記事項 |
---|
ICC/IF
Use a concentration of 1 - 5 µg/ml. |
IHC-P
Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. |
WB
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
Use at an assay dependent concentration. |
ターゲット情報
-
機能
NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1. -
配列類似性
Contains 1 RHD (Rel-like) domain. -
ドメイン
the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors. -
翻訳後修飾
Ubiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'. -
細胞内局在
Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction. - Information by UniProt
-
参照データベース
- Entrez Gene: 5970 Human
- Entrez Gene: 19697 Mouse
- Entrez Gene: 309165 Rat
- Omim: 164014 Human
- SwissProt: Q04206 Human
- SwissProt: Q04207 Mouse
- Unigene: 502875 Human
- Unigene: 249966 Mouse
-
別名
- Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody
- MGC131774 antibody
- NF kappa B p65delta3 antibody
see all
画像
-
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 kDaLane 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 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.
-
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).
-
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) 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) 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.
-
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.
-
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 minutesThis 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.
プロトコール
データシートおよび資料
-
SDS download
-
Datasheet download
参考文献 (1222)
ab16502 は 1222 報の論文で使用されています。
- An Q et al. MCPIP1 alleviates depressive‑like behaviors in mice by inhibiting the TLR4/TRAF6/NF‑κB pathway to suppress neuroinflammation. Mol Med Rep 29:N/A (2024). PubMed: 37975259
- Jiang H et al. Acupuncture Ameliorates Depression-Like Behaviors Through Modulating the Neuroinflammation Mediated by TLR4 Signaling Pathway in Rats Exposed to Chronic Restraint Stress. Mol Neurobiol 61:2606-2619 (2024). PubMed: 37917302
- Wei Z et al. Inhibition of secretory leukocyte protease inhibitor (SLPI) promotes the PUMA-mediated apoptosis and chemosensitivity to cisplatin in colorectal cancer cells. Discov Oncol 14:1 (2023). PubMed: 36595102
- He Y et al. The role of annexin A1 peptide in regulating PI3K/Akt signaling pathway to reduce lung injury after cardiopulmonary bypass in rats. Perfusion 38:320-329 (2023). PubMed: 34951334
- Gao J et al. Icariside II preconditioning evokes robust neuroprotection against ischaemic stroke, by targeting Nrf2 and the OXPHOS/NF-κB/ferroptosis pathway. Br J Pharmacol 180:308-329 (2023). PubMed: 36166825