| 產(chǎn)品編號 | bs-5662R |
| 英文名稱 | phospho-NFKB p65 (Thr254) Rabbit pAb |
| 中文名稱 | 磷酸化細胞核因子抗體 |
| 別 名 | TF65_HUMAN; Transcription factor p65; RELA; Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3; NFKB3; RELA proto-oncogene, NF-kB subunit; p65; CMCU; NFKB3; AIF3BL3; NF-kB p65/RelA; NFκB-p65; NFκB p65; NF κB-p65; NFκBp65; |
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Specific References (3) | bs-5662R has been referenced in 3 publications.
[IF=5.162] Lei Zhao. et al. Proteomic analysis reveals the molecular mechanism of Hippophae rhamnoides polysaccharide intervention in LPS-induced inflammation of IPEC-J2 cells in piglets. Int J Biol Macromol. 2020 Dec;164:3294 WB ; Pig.
[IF=3.889] Ming Dong Si. et al. Swertia mussotii prevents high-fat diet-induced non-alcoholic fatty liver disease in rats by inhibiting expression the TLR4/MyD88 and the phosphorylation of NF-κB. PHARM BIOL. 2022 Oct 07 WB ; Rat.
[IF=3.471] Xiaoyu Wang. et al. Two kinds of traditional Chinese medicine prescriptions reduce thymic inflammation levels and improve humoral immunity of finishing pigs.. FRONT VET SCI. 2022 Sep;9:929112-929112 WB ; Pig.
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| 產(chǎn)品類型 | 磷酸化抗體 |
| 研究領(lǐng)域 | 腫瘤 免疫學(xué) 染色質(zhì)和核信號 信號轉(zhuǎn)導(dǎo) 轉(zhuǎn)錄調(diào)節(jié)因子 激酶和磷酸酶 |
| 抗體來源 | Rabbit |
| 克隆類型 | Polyclonal |
| 克 隆 號 | |
| 交叉反應(yīng) | Human,Mouse,Rat (predicted: Pig,Cow,Dog) |
| 產(chǎn)品應(yīng)用 | WB=1:500-2000,IHC-P=1:100-500,IHC-F=1:100-500,IF=1:100-500,Flow-Cyt=2ug/Test
not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. |
| 理論分子量 | 61 kDa |
| 檢測分子量 | |
| 細胞定位 | 細胞核 細胞漿 |
| 性 狀 | Liquid |
| 濃 度 | 1mg/ml |
| 免 疫 原 | KLH conjugated Synthesised phosphopeptide derived from human NFKBp65 around the phosphorylation site of Thr254: FR(p-T)PP |
| 亞 型 | IgG |
| 純化方法 | affinity purified by Protein A |
| 緩 沖 液 | 0.01M TBS (pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol. |
| 保存條件 | Shipped at 4℃. Store at -20℃ for one year. Avoid repeated freeze/thaw cycles. |
| 注意事項 | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications. |
| PubMed | PubMed |
| 產(chǎn)品介紹 |
NF-kappa-B is a ubiquitous transcription factor involved in several biological processes. It is held in the cytoplasm in an inactive state by specific inhibitors. Upon degradation of the inhibitor, NF-kappa-B moves to the nucleus and activates transcription of specific genes. NF-kappa-B is composed of NFKB1 or NFKB2 bound to either REL, RELA, or RELB. The most abundant form of NF-kappa-B is NFKB1 complexed with the product of this gene, RELA. Four transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]. Function: NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes 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. Subunit: Component of the NF-kappa-B p65-p50 complex. Component of the NF-kappa-B p65-c-Rel complex. Homodimer; component of the NF-kappa-B p65-p65 complex. Component of the NF-kappa-B p65-p52 complex. May interact with ETHE1. Binds AES and TLE1. Interacts with TP53BP2. Binds to and is phosphorylated by the activated form of either RPS6KA4 or RPS6KA5. Interacts with ING4 and this interaction may be indirect. Interacts with CARM1, USP48 and UNC5CL. Interacts with IRAK1BP1 (By similarity). Interacts with NFKBID (By similarity). Interacts with NFKBIA. Interacts with GSK3B. Interacts with NFKBIB (By similarity). Interacts with NFKBIE. Interacts with NFKBIZ. Interacts with EHMT1 (via ANK repeats) (By similarity). Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, IKBKAP and MAP3K14. Interacts with HDAC3; HDAC3 mediates the deacetylation of RELA. Interacts with HDAC1; the interaction requires non-phosphorylated RELA. Interacts with CBP; the interaction requires phosphorylated RELA. Interacts (phosphorylated at 'Thr-254') with PIN1; the interaction inhibits p65 binding to NFKBIA. Interacts with SOCS1. Interacts with UXT. Interacts with MTDH and PHF11. Interacts with ARRB2. Interacts with human respiratory syncytial virus (HRSV) protein M2-1. Interacts with NFKBIA (when phosphorylated), the interaction is direct; phosphorylated NFKBIA is part of a SCF(BTRC)-like complex lacking CUL1. Interacts with RNF25. Interacts (via C-terminus) with DDX1. Interacts with UFL1 and COMMD1. Interacts with BRMS1; this promotes deacetylation of 'Lys-310'. Interacts with NOTCH2 (By similarity). Directly interacts with MEN1; this interaction represses NFKB-mediated transactivation. Interacts with AKIP1, which promotes the phosphorylation and nuclear retention of RELA. Interacts (via the RHD) with GFI1; the interaction, after bacterial lipopolysaccharide (LPS) stimulation, inhibits the transcriptional activity by interfering with the DNA-binding activity to target gene promoter DNA. Subcellular Location: Nucleus. Cytoplasm. Note=Colocalized with DDX1 in the nucleus upon TNF-alpha induction. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalizes with GFI1 in the nucleus after LPS stimulation. Post-translational modifications: 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. Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity. Phosphorylation at Ser-276 by RPS6KA4 and RPS6KA5 promotes its transactivation and transcriptional activities. Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3 and SIRT2. 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. Lys-310 is deacetylated by SIRT2. S-nitrosylation of Cys-38 inactivates the enzyme activity. Sulfhydration at Cys-38 mediates the anti-apoptotic activity by promoting the interaction with RPS3 and activating the transcription factor activity. Sumoylation by PIAS3 negatively regulates DNA-bound activated NF-kappa-B. Similarity: Contains 1 RHD (Rel-like) domain. SWISS: Q04206 Gene ID: 5970 Database links: Entrez Gene: 5970 Human Omim: 164014 Human SwissProt: Q04206 Human Unigene: 502875 Human NF-κBp65是一種重要的轉(zhuǎn)錄因子，NF-kBp65可激活參與炎癥、細胞增殖、細胞凋亡等基因的調(diào)節(jié)，影響著細胞的凋亡，同時影響著腫瘤細胞對細胞毒性藥物及離子輻射的敏感性。ras基因誘導(dǎo)的致癌突變作用需NFkB的活化，提示NFkB在致癌發(fā)生方面可能起一定作用；另有文獻報道，在乳腺癌、非小細胞性肺癌、甲狀腺癌、T或B淋巴細胞白血病及病毒誘變導(dǎo)致的腫瘤等人類腫瘤中，NFkB活化或表達。 NF-кB可以保護細胞免受腫瘤壞死因子以及電離輻射等引起的凋亡作用，而抑制NFkB的表達可以增加TNF等引起的細胞凋亡，以及增加化療及放療對腫瘤細胞的敏感性。 |
| 產(chǎn)品圖片 |
Sample:
Lung (Mouse) Lysate at 40 ug
Spleen (Mouse) Lysate at 40 ug
Primary: Anti- phospho-NFKB p65(Thr254) (bs-5662R) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution
Predicted band size: 61 kD
Observed band size: 61 kD
Paraformaldehyde-fixed, paraffin embedded (Human stomach carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-NFKB p65(Thr254)) Polyclonal Antibody, Unconjugated (bs-5662R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human cervical carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-NFKB p65(Thr254)) Polyclonal Antibody, Unconjugated (bs-5662R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-NFKB p65(Thr254)) Polyclonal Antibody, Unconjugated (bs-5662R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Blank control:Mouse spleen.
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Thr254) antibody (bs-5662R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature.Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control:A431.
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Thr254) antibody (bs-5662R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Overlay histogram showing HL 60 cells stained with bs-5662R (Green line). The cells were fixed with 90% methanol (5 min) and then permeabilized with 0.01M PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (bs-5662R,3μg/1x10^6 cells) for 30 min at 22℃. The secondary antibody used was fluorescein isothiocyanate goat anti-rabbit IgG (H+L) (bs- 0295G-FITC , Brillant blue line) at 1/200 dilution for 30 min at 22℃. Isotype control antibody was rabbit IgG (polyclonal,bs-0295P,Orange line) (3μg/1x10^6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of 20,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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| 1、抗體溶解方法 | |
| 2、抗體修復(fù)方式 | |
| 3、常用試劑的配制 | |
| 4、免疫組化操作步驟 | |
| 5、免疫組化問題解答 | |
| 6、Western Blotting 操作步驟 | |
| 7、Western Blotting 問題解答 | |
| 8、關(guān)于肽鏈的設(shè)計 | |
| 9、多肽的溶解與保存 | |
| 10、酶標抗體效價測定程序 | |
