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Rabbit Anti-phospho-NFKB p65 (Ser536)  antibody (bs-0982R)
~~~促销,代码KX240301~~~
~~~促销,代码KX240302~~~
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说明书: 50ul  100ul  200ul
50ul/1180.00元
100ul/1980.00元
200ul/2800.00元
大包装/询价

产品编号 bs-0982R
英文名称 phospho-NFKB p65 (Ser536)
中文名称 磷酸化细胞核因子抗体
别    名 NF-kB p65 (phospho S536); p-NF-κB p65(Phospho-Ser536); RELA(phospho S536); NF kB P65; NF-kB p65; NFKBp65; NF-κBp65; p65 NF kappaB; p65 NFkB; NFKBp65; RELA; Transcription Factor p65; v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)); V Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A; v rel reticuloendotheliosis viral oncogene homolog A (avian); v-rel reticuloendotheliosis viral oncogene homolog A; p65NFKB; Avian reticuloendotheliosis viral (v rel) oncogene homolog A; MGC131774; NFKB 3; NFKB3; Nuclear Factor NF Kappa B p65 Subunit; Nuclear factor of kappa light polypeptide gene enhancer in B cells 3; Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B Cells; TF65_HUMAN.  NFκB-p65; NFκB p65; NF κB-p65; NFκBp65;
Specific References  (115)     |     bs-0982R has been referenced in 115 publications.
[IF=17.521] Yi Yan. et al. Nanomedicines Reprogram Synovial Macrophages by Scavenging Nitric Oxide and Silencing CA9 in Progressive Osteoarthritis. Advanced Science. 2023 Feb;:2207490  WB ;  Mouse.  
[IF=15.304] Sitong Liu. et al. MRI-visible mesoporous polydopamine nanoparticles with enhanced antioxidant capacity for osteoarthritis therapy. BIOMATERIALS. 2023 Apr;295:122030  WB ;  Mouse.  
[IF=10.684] Chen Zhang. et al. The novel hyaluronic acid granular hydrogel attenuates osteoarthritis progression by inhibiting the TLR-2/NF-κB signaling pathway through suppressing cellular senescence. BIOENG TRANSL MED. 2022 Dec;:e10475  WB ;  Mouse.  
[IF=10.435] Xu, Hua-Zhen. et al. Synergy of nanodiamond–doxorubicin conjugates and PD-L1 blockade effectively turns tumor-associated macrophages against tumor cells. J Nanobiotechnol. 2021 Dec;19(1):1-24  IHC ;  mouse.  
[IF=10.372] Zhang Qingzhuo. et al. Phenylalanine diminishes M1 macrophage inflammation. SCI CHINA LIFE SCI. 2023 May;:1-15  WB ;  Mouse.  
[IF=9.995] Jian Fu. et al. GABA regulates IL-1β production in macrophages. CELL REP. 2022 Dec;41:111770  WB ;  Mouse.  
[IF=9.038] Xuting Liu. et al. Amorphous silica nanoparticles induce inflammation via activation of NLRP3 inflammasome and HMGB1/TLR4/MYD88/NF-kb signaling pathway in HUVEC cells. J Hazard Mater. 2021 Feb;404:124050  WB ;  Human.  
[IF=7.59] Muzhe Li. et al. STS load PCL- MECM based hydrogel hybrid scaffold promote meniscal regeneration via modulating macrophage phenotype polarization. BIOMATER SCI-UK. 2023 Jan;:  WB ;  Rabbit.  
[IF=7.561] Hao Wang. et al. Aspartate Metabolism Facilitates IL-1β Production in Inflammatory Macrophages. Front Immunol. 2021; 12: 753092  WB ;  Mouse.  
[IF=7.419] Lingli Sun. et al. Extract of Jasminum grandiflorum L. alleviates CCl4-induced liver injury by decreasing inflammation, oxidative stress and hepatic CYP2E1 expression in mice. BIOMED PHARMACOTHER. 2022 Aug;152:113255  IHC ;  Mouse.  
[IF=7.31] Peng Ge. et al. Ferroptosis in Rat Lung Tissue during Severe Acute Pancreatitis-Associated Acute Lung Injury: Protection of Qingyi Decoction. OXID MED CELL LONGEV. 2023;2023:5827613  WB ;  Rat.  
[IF=6.993] Xiaoyue Guan. et al. Gremlin aggravates periodontitis via activating the NF-κB signaling pathway. 2022 Jan 06  IF,IHC ;  Human.  
[IF=6.706] Na Chen. et al. Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation. NUTRIENTS. 2022 Jan;14(23):5067  WB ;  Rat.  
[IF=6.529] Yizi Zhang. et al. Gastroprotective effects of extract of Jasminum grandiflorum L. flower in HCl/EtOH-induced gastric mucosal ulceration mice. Biomed Pharmacother. 2021 Dec;144:112268  IHC ;  Mouse.  
[IF=6.291] Sihong Li. et al. Protective role of curcumin on aflatoxin B1-induced TLR4/RIPK pathway mediated-necroptosis and inflammation in chicken liver. Ecotox Environ Safe. 2022 Mar;233:113319  WB ;  Chicken.  
[IF=6.291] Shaofeng Wu. et al. The neuroprotective effect of curcumin against ATO triggered neurotoxicity through Nrf2 and NF-κB signaling pathway in the brain of ducks. Ecotox Environ Safe. 2021 Dec;228:112965  WB ;  Duck.  
[IF=6.117] Yuanyuan Xing. et al. Artemisia ordosica polysaccharide ameliorated LPS-induced growth inhibition and intestinal injury in broilers through enhancing immune-regulation and antioxidant capacity. J NUTR BIOCHEM. 2023 Feb;:109284  WB ;  Chicken.  
[IF=6.005] Yishan Zhou. et al. Neferine Suppresses Experimental Colitis-Associated Colorectal Cancer by Inhibition of NF-κB p65 and STAT3. AM J CHINESE MED. 2022 Jun 22  WB ;  Mouse.  
[IF=5.988] Liu-Gen Li. et al. Dihydroartemisinin remodels macrophage into an M1 phenotype via ferroptosis-mediated DNA damage. FRONT PHARMACOL. 2022; 13: 949835  WB ;  Mouse.  
[IF=5.984] Amitha Muraleedharan. et al. Protein kinase C eta is activated in reactive astrocytes of an Alzheimer's disease mouse model: Evidence for its immunoregulatory function in primary astrocytes. Glia. 2021 Mar;69(3):697-714  WB ;  Mouse.  
[IF=5.81] Yu TT. et al. Chlorin e6-Induced Photodynamic Effect Polarizes the Macrophage Into an M1 Phenotype Through Oxidative DNA Damage and Activation of STING.. Front Pharmacol. 2022 Mar;13:837784-837784  WB ;  Mouse.  
[IF=5.81] Xu X. et al. Alhagi pseudalhagi Extract Exerts Protective Effects Against Intestinal Inflammation in Ulcerative Colitis by Affecting TLR4-Dependent NF-κB Signaling Pathways.. Front Pharmacol. 2021 Nov;12:764602-764602  WB ;  Mouse.  
[IF=5.717] Na Chen. et al. Casein Oligochitosan-Glycation by Transglutaminase Enhances the Anti-Inflammatory Potential of Casein Hydrolysates to the Lipopolysaccharide-Stimulated IEC-6 Cells. Nutrients. 2022 Jan;14(3):686  WB ;  Rat.  
[IF=5.34] Dian-Dong Hou. et al. Therapeutic effects of myricetin on atopic dermatitis in vivo and in vitro. PHYTOMEDICINE. 2022 Jul;102:154200  WB ;  Mouse.  
[IF=5.265] Li, Ke. et al. The Prevention Effect of Lactobacillus plantarum 17–5 on Escherichia coli-Induced Mastitis in Mice. PROBIOTICS ANTIMICRO. 2023 Feb;:1-9  WB ;  Mouse.  
[IF=5.235] Pu, Luya. et al. Laminar shear stress alleviates monocyte adhesion and atherosclerosis development via miR-29b-3p/CX3CL1 axis regulation. J CELL SCI. 2022 Jul;135(14):  WB ;  Human.  
[IF=5.195] Ming-Hui Li. et al. Fufang Zhenzhu Tiaozhi capsule ameliorates hyperuricemic nephropathy by inhibition of PI3K/AKT/NF-κB pathway. J ETHNOPHARMACOL. 2022 Aug;:115644  WB ;  Mouse.  
[IF=5.19] Tang, Lixuan. et al. Curcumin antagonizes inflammation and autophagy induced by arsenic trioxide through immune protection in duck spleen. ENVIRON SCI POLLUT R. 2022 Jun;:1-12  WB ;  Duck.  
[IF=5.156] Li, Muzhe. et al. Effects of adenovirus-mediated knockdown of IRAK4 on synovitis in the osteoarthritis rabbit model. Arthritis Res Ther. 2021 Dec;23(1):1-12  WB ;  Rabbits.  
[IF=5.085] Chen Siyuan. et al. Serine Supports IL-1β Production in Macrophages Through mTOR Signaling. Front Immunol. 2020 Aug;11:1866  WB ;  Mouse.  
产品类型 磷酸化抗体 
研究领域 肿瘤  染色质和核信号  信号转导  转录调节因子  激酶和磷酸酶  
抗体来源 Rabbit
克隆类型 Polyclonal
交叉反应 Human,Mouse,Rat (predicted: Chicken,Dog,Cow,Horse)
产品应用 WB=1:500-2000, IHC-P=1:100-500, Flow-Cyt=1μg /test
not yet tested in other applications.
optimal dilutions/concentrations should be determined by the end user.
理论分子量 61kDa
细胞定位 细胞核 细胞浆 
性    状 Liquid
浓    度 1mg/ml
免 疫 原 KLH conjugated Synthesised phosphopeptide derived from human NFKBp65 around the phosphorylation site of Ser536: FS(p-S)IA 
亚    型 IgG
纯化方法 affinity purified by Protein A
缓 冲 液 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol.
保存条件 Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.
注意事项 This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications.
PubMed PubMed
产品介绍 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

Entrez Gene: 19697 Mouse

Entrez Gene: 309165 Rat

Omim: 164014 Human

SwissProt: Q04206 Human

SwissProt: Q04207 Mouse

Unigene: 502875 Human

Unigene: 249966 Mouse

Unigene: 19480 Rat



转录调节因子(Transcriptin Regulators)
NF-κBp65是一种重要的转录因子,NF-kBp65可激活参与炎症、细胞增殖、细胞凋亡等基因的调节,影响着细胞的凋亡,同时影响着肿瘤细胞对细胞毒性药物及离子辐射的敏感性。ras基因诱导的致癌突变作用需NFkB的活化,提示NFkB在致癌发生方面可能起一定作用;另有文献报道,在乳腺癌、非小细胞性肺癌、甲状腺癌、T或B淋巴细胞白血病及病毒诱变导致的肿瘤等人类肿瘤中,NFkB活化或表达。
NF-кB可以保护细胞免受肿瘤坏死因子以及电离辐射等引起的凋亡作用,而抑制NFkB的表达可以增加TNF等引起的细胞凋亡,以及增加化疗及放疗对肿瘤细胞的敏感性。
产品图片
Sample:
Lane 1: SiHa (Human) Cell Lysate at 30 ug
Lane 2: MCF-7 (Human) Cell Lysate at 30 ug
Lane 3: A431 (Human) Cell Lysate at 30 ug
Primary: Anti-phospho-NFKB p65 (Ser536) (bs-0982R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 62 kD
Sample:
Lane 1: Large intestine (Mouse) Lysate at 40 ug
Lane 2: Large intestine (Rat) Lysate at 40 ug
Lane 3: Lung (Mouse) Lysate at 40 ug
Lane 4: Lung (Rat) Lysate at 40 ug
Lane 5: Bladder (Mouse) Lysate at 40 ug
Lane 6: RAW264.7 (Mouse) Cell Lysate at 30 ug
Primary: Anti-phospho-NFKB p65 (Ser536) (bs-0982R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 62 kD
Sample:
Lane 1: MCF-7 (Human) Cell Lysate at 30 ug
Lane 2: A431 (Human) Cell Lysate at 30 ug
Lane 3: HL60 (Human) Cell Lysate at 30 ug
Lane 4: Raw264.7 (Mouse) Cell Lysate at 30 ug
Lane 5: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 6: Jurkat (Human) Cell Lysate at 30 ug
Lane 7: A549 (Human) Cell Lysate at 30 ug
Primary: Anti-phospho-NFKB p65 (Ser536) (bs-0982R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 65 kD
Sample:
Molt-4(Human) Cell Lysate at 30 ug
A549(Human) Cell Lysate at 30 ug
Jurkat(Human) Cell Lysate at 30 ug
Primary: Anti-phospho-NFKB p65 (Ser536) (bs-0982R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 65 kD
Observed band size: 62 kD
Sample:
Lung (Mouse) Lysate at 40 ug
Primary: Anti-phospho-NFKB p65(Ser536) (bs-0982R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 61 kD
Observed band size: 61 kD
Paraformaldehyde-fixed, paraffin embedded (human breast); 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) Polyclonal Antibody, Unconjugated (bs-0982R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human pancreatic cancer); 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 (Ser536)) Polyclonal Antibody, Unconjugated (bs-0982R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse bladder); 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 (Ser536)) Polyclonal Antibody, Unconjugated (bs-0982R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat spleen); 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 (Ser536)) Polyclonal Antibody, Unconjugated (bs-0982R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat uterus); 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 (Ser536)) Polyclonal Antibody, Unconjugated (bs-0982R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Blank control (blue line): HL60 (blue).
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65(Ser536) antibody (bs-0982R),Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC, Dilution: 1μg /test.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control (blue line): Hela (blue).
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65(Ser536) antibody (bs-0982R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control: HL-60.
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Ser536) antibody (bs-0982R)
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.
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