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Rabbit Anti-Phospho-P38 MAPK (Thr180 + Tyr182)  antibody (bs-0636R)  
~~~促销代码KT202411~~~
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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-0636R
英文名称 Rabbit Anti-Phospho-P38 MAPK (Thr180 + Tyr182)  antibody
中文名称 磷酸化-丝裂原活化蛋白激酶p38抗体(P-p38 MAPK)
别    名 P38 MAPK(Phospho-Thr180); phospho-MAPK14(Thr180/Tyr182); MAPK14(phospho Thr180/Tyr182); CSAID Binding Protein 1; CSAID binding protein; CSAID-binding protein; Csaids binding protein; CSBP 1; CSBP 2; CSBP; CSBP1; CSBP2; CSPB 1; CSPB1; Cytokine suppressive anti inflammatory drug binding protein; Cytokine suppressive anti-inflammatory drug-binding protein; EXIP; MAP kinase 14; MAP kinase MXI2; MAP kinase p38 alpha; MAPK 14; MAPK14; MAX interacting protein 2; MAX-interacting protein 2; Mitogen Activated Protein Kinase 14; Mitogen activated protein kinase p38 alpha; Mitogen-activated protein kinase 14; Mitogen-activated protein kinase p38 alpha; MK14_HUMAN; Mxi 2; Mxi2; p38 ALPHA; p38; p38 MAP kinase; p38 MAPK; p38 mitogen activated protein kinase; p38ALPHA; p38alpha Exip; PRKM14; PRKM15; RK; SAPK 2A; SAPK2A; Stress Activated Protein Kinase 2A.  
Specific References  (41)     |     bs-0636R has been referenced in 41 publications.
[IF=20.693] Myung-Ju Lee. et al. CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation. J MED VIROL. 2022 Jul;:  WB ;  Human.  
[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=7.675] Honghong Zhan. et al. Oxybaphus himalaicus Mitigates Lipopolysaccharide-Induced Acute Kidney Injury by Inhibiting TLR4/MD2 Complex Formation. ANTIOXIDANTS-BASEL. 2022 Dec;11(12):2307  WB ;  Mouse.  
[IF=7.129] Jin Chen. et al. Surface functionalization-dependent inflammatory potential of polystyrene nanoplastics through the activation of MAPK/ NF-κB signaling pathways in macrophage Raw 264.7. ECOTOX ENVIRON SAFE. 2023 Feb;251:114520  WB ;  Mouse.  
[IF=7.129] Li Xu. et al. Fenpropathrin increases gliquidone absorption via causing damage to the integrity of intestinal barrier. ECOTOX ENVIRON SAFE. 2022 Sep;242:113882  WB ;  Rat.  
[IF=5.923] Junfeng Ke. et al. CTI-2 Inhibits Metastasis and Epithelial-Mesenchymal Transition of Breast Cancer Cells by Modulating MAPK Signaling Pathway. Int J Mol Sci. 2021 Jan;22(22):12229  WB,IF ;  Human.  
[IF=5.614] Zhu J et al. SPARC Promotes Self‐Renewal of Limbal Epithelial Stem Cells and Ocular Surface Restoration through JNK and p38‐MAPK Signaling Pathways. Stem Cells. 2019 Oct 23.  WB ;  Rabbit.  
[IF=5.455] Zhang, Rongrong. et al. Compound traditional Chinese medicine dermatitis ointment ameliorates inflammatory responses and dysregulation of itch-related molecules in atopic dermatitis. Chin Med-Uk. 2022 Dec;17(1):1-19  WB ;  Mouse.  
[IF=5.307] Xiaobao Gong. et al. Synthesis and anti-inflammatory activity of paeonol derivatives with etherized aryl urea by regulating TLR4/MyD88 signaling pathway in RAW264.7 cell. BIOORG CHEM. 2022 Oct;127:105939  WB ;  Mouse.  
[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=4.996] Cong Changsheng. et al. Renin-angiotensin system inhibitors mitigate radiation pneumonitis by activating ACE2-angiotensin-(1–7) axis via NF-κB/MAPK pathway. SCI REP-UK. 2023 May;13(1):1-11  WB ;  Mouse.  
[IF=4.432] Chao Li. et al. Research on the effect and underlying molecular mechanism of Cangzhu in the treatment of gouty arthritis. EUR J PHARMACOL. 2022 Jul;927:175044  WB ;  Mouse.  
[IF=4.411] Yudan Zhao. et al. COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis. PLOS NEGLECT TROP D. 2022 Apr;16(4):e0010402  WB ;  Mouse.  
[IF=4.407] Yaping Wang. et al. Blockade of GITRL/GITR signaling pathway attenuates house dust mite-induced allergic asthma in mice through inhibition of MAPKs and NF-κB signaling. Mol Immunol. 2021 Sep;137:238  IF ;  Mouse.  
[IF=4.315] Ma, Dufang. et al. Astragalus polysaccharide prevents heart failure-induced cachexia by alleviating excessive adipose expenditure in white and brown adipose tissue. LIPIDS HEALTH DIS. 2023 Dec;22(1):1-16  WB ;  Rat.  
[IF=4.26] Rosenzweig, Derek H., et al. "Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis." Osteoarthritis and Cartilage (2012).  WB ;  Bovine.  
[IF=4.171] Yan Zhang. et al. Polysaccharide from Ganoderma lucidum ameliorates cognitive impairment by regulating the inflammation of the brain–liver axis in rats. 2021 May 18  WB ;  Rat.  
[IF=4.146] Jian Kang. et al. Ginsenoside Rg1 Mitigates Porcine Intestinal Tight Junction Disruptions Induced by LPS through the p38 MAPK/NLRP3 Inflammasome Pathway. TOXICS. 2022 Jun;10(6):285  WB ;  Mouse,Pig.  
[IF=3.943] Xiuhong Wang. et al. Protective effect of combination of anakinra and MCC950 against acute lung injury is achieved through suppression of the NF-κB-mediated-MAPK and NLRP3-caspase pathways. Int Immunopharmacol. 2021 Aug;97:107506  WB ;  Mouse.  
[IF=3.921] Meiyun Cai. et al. Kaemperfol alleviates pyroptosis and microglia-mediated neuroinflammation in Parkinson's disease via inhibiting p38MAPK/NF-κB signaling pathway. Neurochem Int. 2021 Nov;:105221  WB ;  Rat.  
[IF=3.766] Song G et al. Chrysophanol attenuates airway inflammation and remodeling through nuclear factor‐kappa B signaling pathway in asthma. Phytother Res. 2019 Jul 17.  WB ;  Mouse&Human.  
[IF=3.738] Lili Liu. et al. Rutin Ameliorates Cadmium-Induced Necroptosis in the Chicken Liver via Inhibiting Oxidative Stress and MAPK/NF-κB Pathway. 2021 Jun 06  WB ;  Chicken.  
[IF=3.67] Zhao Z et al. Lactobacillus plantarum NA136 ameliorates nonalcoholic fatty liver disease by modulating gut microbiota, improving intestinal barrier integrity, and attenuating inflammation. Appl Microbiol Biotechnol. 2020 Apr 26.  WB ;  Mouse.  
[IF=3.585] Deng Z et al. M1 macrophage mediated increased reactive oxygen species (ROS) influence wound healing via the MAPK signaling in vitro and in vivo. Toxicol Appl Pharmacol. 2019 Mar 1;366:83-95.  IHC-P ;  Human.  
[IF=3.266] Ma Q et al. Vitamin B5 inhibit RANKL induced osteoclastogenesis and ovariectomy induced osteoporosis by scavenging ROS generation. Am J Transl Res. 2019 Aug 15;11(8):5008-5018. eCollection 2019.  WB ;  Mouse.  
[IF=3.118] Fu S et al. Berberine suppresses mast cell-mediated allergic responses via regulating FcɛRI-mediated and MAPK signaling.Int Immunopharmacol. 2019 Jun;71:1-6.  WB ;  Human.  
[IF=3.111] Yinxi Yang. et al. Heat shock protein 20 suppresses breast carcinogenesis by inhibiting the MAPK and AKT signaling pathways. ONCOL LETT. 2022 Dec;24(6):1-11  WB ;  Human.  
[IF=3.098] Qiumei Shi. et al. Echinacea polysaccharide attenuates lipopolysaccharide‑induced acute kidney injury via inhibiting inflammation, oxidative stress and the MAPK signaling pathway. Int J Mol Med. 2021 Jan;47(1):243-255  IF ;  Rat.  
[IF=2.952] Linyan Cheng. et al. Icariin attenuates thioacetamide‑induced bone loss via the RANKL‑p38/ERK‑NFAT signaling pathway. Mol Med Rep. 2022 Apr;25(4):1-11  WB ;  Rat.  
[IF=2.942] Liu A et al. Cimifugin ameliorates imiquimod-induced psoriasis by inhibiting oxidative stress and inflammation via NF-κB/MAPK pathway. Biosci Rep. 2020 Jun 26;40(6):BSR20200471.  WB ;  Mouse&Human.  
产品类型 磷酸化抗体 
研究领域 肿瘤  细胞生物  免疫学  信号转导  细胞凋亡  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: Rabbit,Dog)
产品应用 WB=1:500-2000,IHC-P=1:100-500,IHC-F=1:100-500,Flow-Cyt=1μg/Test,IF=1:100-500
not yet tested in other applications.
optimal dilutions/concentrations should be determined by the end user.
理论分子量 42kDa
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 p38 MAPK around the phosphorylation site of Thr180/Tyr182: M(p-T)G(p-Y)VA 
亚    型 IgG
纯化方法 affinity purified by Protein A
缓 冲 液 0.01M TBS (pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
保存条件 Shipped at 4℃. Store at -20℃ 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 The protein encoded by this gene is a member of the MAP kinase family. MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. This kinase is activated by various environmental stresses and proinflammatory cytokines. The activation requires its phosphorylation by MAP kinase kinases(MKKs), or its autophosphorylation triggered by the interaction of MAP3K7IP1/TAB1 protein with this kinase. The substrates of this kinase include transcription regulator ATF2, MEF2C, and MAX, cell cycle regulator CDC25B, and tumor suppressor p53, which suggest the roles of this kinase in stress related transcription and cell cycle regulation, as well as in genotoxic stress response. Four alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported.

Function:
Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. MAPK14 is one of the four p38 MAPKs which play an important role in the cascades of cellular responses evoked by extracellular stimuli such as proinflammatory cytokines or physical stress leading to direct activation of transcription factors. Accordingly, p38 MAPKs phosphorylate a broad range of proteins and it has been estimated that they may have approximately 200 to 300 substrates each. Some of the targets are downstream kinases which are activated through phosphorylation and further phosphorylate additional targets. RPS6KA5/MSK1 and RPS6KA4/MSK2 can directly phosphorylate and activate transcription factors such as CREB1, ATF1, the NF-kappa-B isoform RELA/NFKB3, STAT1 and STAT3, but can also phosphorylate histone H3 and the nucleosomal protein HMGN1. RPS6KA5/MSK1 and RPS6KA4/MSK2 play important roles in the rapid induction of immediate-early genes in response to stress or mitogenic stimuli, either by inducing chromatin remodeling or by recruiting the transcription machinery. On the other hand, two other kinase targets, MAPKAPK2/MK2 and MAPKAPK3/MK3, participate in the control of gene expression mostly at the post-transcriptional level, by phosphorylating ZFP36 (tristetraprolin) and ELAVL1, and by regulating EEF2K, which is important for the elongation of mRNA during translation. MKNK1/MNK1 and MKNK2/MNK2, two other kinases activated by p38 MAPKs, regulate protein synthesis by phosphorylating the initiation factor EIF4E2. MAPK14 interacts also with casein kinase II, leading to its activation through autophosphorylation and further phosphorylation of TP53/p53. In the cytoplasm, the p38 MAPK pathway is an important regulator of protein turnover. For example, CFLAR is an inhibitor of TNF-induced apoptosis whose proteasome-mediated degradation is regulated by p38 MAPK phosphorylation. In a similar way, MAPK14 phosphorylates the ubiquitin ligase SIAH2, regulating its activity towards EGLN3. MAPK14 may also inhibit the lysosomal degradation pathway of autophagy by interfering with the intracellular trafficking of the transmembrane protein ATG9. Another function of MAPK14 is to regulate the endocytosis of membrane receptors by different mechanisms that impinge on the small GTPase RAB5A. In addition, clathrin-mediated EGFR internalization induced by inflammatory cytokines and UV irradiation depends on MAPK14-mediated phosphorylation of EGFR itself as well as of RAB5A effectors. Ectodomain shedding of transmembrane proteins is regulated by p38 MAPKs as well. In response to inflammatory stimuli, p38 MAPKs phosphorylate the membrane-associated metalloprotease ADAM17. Such phosphorylation is required for ADAM17-mediated ectodomain shedding of TGF-alpha family ligands, which results in the activation of EGFR signaling and cell proliferation. Another p38 MAPK substrate is FGFR1. FGFR1 can be translocated from the extracellular space into the cytosol and nucleus of target cells, and regulates processes such as rRNA synthesis and cell growth. FGFR1 translocation requires p38 MAPK activation. In the nucleus, many transcription factors are phosphorylated and activated by p38 MAPKs in response to different stimuli. Classical examples include ATF1, ATF2, ATF6, ELK1, PTPRH, DDIT3, TP53/p53 and MEF2C and MEF2A. The p38 MAPKs are emerging as important modulators of gene expression by regulating chromatin modifiers and remodelers. The promoters of several genes involved in the inflammatory response, such as IL6, IL8 and IL12B, display a p38 MAPK-dependent enrichment of histone H3 phosphorylation on 'Ser-10' (H3S10ph) in LPS-stimulated myeloid cells. This phosphorylation enhances the accessibility of the cryptic NF-kappa-B-binding sites marking promoters for increased NF-kappa-B recruitment. Phosphorylates CDC25B and CDC25C which is required for binding to 14-3-3 proteins and leads to initiation of a G2 delay after ultraviolet radiation. Phosphorylates TIAR following DNA damage, releasing TIAR from GADD45A mRNA and preventing mRNA degradation. The p38 MAPKs may also have kinase-independent roles, which are thought to be due to the binding to targets in the absence of phosphorylation. Protein O-Glc-N-acylation catalyzed by the OGT is regulated by MAPK14, and, although OGT does not seem to be phosphorylated by MAPK14, their interaction increases upon MAPK14 activation induced by glucose deprivation. This interaction may regulate OGT activity by recruiting it to specific targets such as neurofilament H, stimulating its O-Glc-N-acylation. Required in mid-fetal development for the growth of embryo-derived blood vessels in the labyrinth layer of the placenta. Also plays an essential role in developmental and stress-induced erythropoiesis, through regulation of EPO gene expression. Isoform MXI2 activation is stimulated by mitogens and oxidative stress and only poorly phosphorylates ELK1 and ATF2. Isoform EXIP may play a role in the early onset of apoptosis. Phosphorylates S100A9 at 'Thr-113'.

Subunit:
Binds to a kinase interaction motif within the protein tyrosine phosphatase, PTPRR (By similarity). This interaction retains MAPK14 in the cytoplasm and prevents nuclear accumulation. Interacts with SPAG9 and GADD45A. Interacts with CDC25B, CDC25C, DUSP1, DUSP10, DUSP16, NP60, FAM48A and TAB1. Interacts with casein kinase II subunits CSNK2A1 and CSNK2B.

Subcellular Location:
Cytoplasm. Nucleus.

Tissue Specificity:
Brain, heart, placenta, pancreas and skeletal muscle. Expressed to a lesser extent in lung, liver and kidney.

Post-translational modifications:
Dually phosphorylated on Thr-180 and Tyr-182 by the MAP2Ks MAP2K3/MKK3, MAP2K4/MKK4 and MAP2K6/MKK6 in response to inflammatory citokines, environmental stress or growth factors, which a ctivates the enzyme. Dual phosphorylation can also be mediated by TAB1-mediated autophosphorylation. TCR engagement in T-cells also leads to Tyr-323 phosphorylation by ZAP70. Dephosphorylated and inactivated by DUPS1, DUSP10 and DUSP16.
Acetylated at Lys-53 and Lys-152 by KAT2B and EP300. Acetylation at Lys-53 increases the affinity for ATP and enhances kinase activity. Lys-53 and Lys-152 are deacetylated by HDAC3.
Ubiquitinated. Ubiquitination leads to degradation by the proteasome pathway.

Similarity:
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily.
Contains 1 protein kinase domain.

SWISS:
Q16539

Gene ID:
1432

Database links:

Entrez Gene: 1432 Human

Entrez Gene: 26416 Mouse

Entrez Gene: 81649 Rat

Entrez Gene: 403856 Dog

GenBank: NM_001315 Human

GenBank: NM_139012 Human

GenBank: NM_011951 Mouse

GenBank: NM_031020 Rat

Omim: 600289 Human

SwissProt: O02812 Dog

SwissProt: Q16539 Human

SwissProt: P47811 Mouse

SwissProt: P70618 Rat

Unigene: 485233 Human

Unigene: 311337 Mouse

Unigene: 88085 Rat



激酶和磷酸酶(Kinases and Phosphatases)
丝裂原活化蛋白激酶p38(p38 MAPK、磷酸化pERK)参与细胞生长、增殖、分化、死亡及细胞间的功能同步等多种生理过程.
P-p38MAPK是丝裂原活化蛋白激酶家族中的成员之一,大量研究显示p38在能量代谢中具有广泛的作用。p38参与脂肪组织、骨骼肌、胰岛细胞和肝脏等组织、器官的能量代谢.分子量:38KDa
p38 MAPK:作为细胞信号传递系统的交汇点,细胞内普遍存在的一条信号转导通路。细胞外的物理应激因子,如高渗透压、热休克、紫外线以及细胞因子、内毒素脂多糖(LPS)等都能激活该途径,诱导细胞内蛋白质合成与分泌、细胞分化及凋亡等生物效应。p38 MAPK还能与细胞内其他信号通路之间相互作用,是细胞内信号传递系统的交汇点或共同通路。p38 MAPK一旦被激活后,可以使一些转录因子如CREB、转录活化因子-1(activating factor-1, ATF-1)、ATF-2及活化蛋白-1(AP-1)等的丝氨酸和苏氨酸位点磷酸化,活化这些转录因子,从而调节目的基因的表达。 p38(丝氨酸位点)磷酸化后可以直接激活转录因子,参与机体的应激反应。
产品图片
Sample: Muscle (Mouse) Lysate at 40 ug Muscle (Rat) Lysate at 40 ug Primary: Anti-Phospho-P38 MAPK (Thr180 + Tyr182) (bs-0636R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 42 kD Observed band size: 42 kD
Sample: Kidney (Mouse) Lysate at 40 ug Primary: Anti-Phospho-P38 MAPK (Thr180 + Tyr182) (bs-0636R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 42 kD Observed band size: 42 kD
Paraformaldehyde-fixed, paraffin embedded (rat brain tissue); 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 (P-P38 MAPK) Polyclonal Antibody, Unconjugated (bs-0636R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: mouse brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (bs-0636R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: human placenta tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (bs-0636R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (bs-0636R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Blank control: HepG2(blue). Primary Antibody:Rabbit Anti-Phospho-P38 MAPK (Thr180 + Tyr182)antibody (bs-0636R,Green); Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions; Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA. Protocol The cells were fixed with 2% paraformaldehyde for 10 min at 37℃. Primary antibody (bs-0636R, 1μg /1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/FITC antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control:MCF7. Primary Antibody (green line): Rabbit Anti-Phospho-P38 MAPK (Thr180 + Tyr182) antibody (bs-0636R) Dilution: 2μg /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 0.1%PBST for 20 min at room temperature. 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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