| Species | Gene ID | Accession | Source | Length | MW | Tag |
| Human | 7124 | P01375 | E. coli | 157aa | 17.3kDa | No |
| 蛋白信息(About this protein) | |
| 名称(Name) | Recombinant Human Soluble TNF-α (Trimeric form)；重组可溶性人肿瘤坏死因子α (三聚体) |
| 别名(Synonyms) | Cachetin; DIF; TNF; tnfa; tnf-a; TNFSF1A; TNFSF2; APC1 protein; Cachectin; DIF; TNF, monocyte-derived; TNF-a; TNF-alphacachectin; TNFATNF, macrophage-derived; TNFSF2TNF superfamily, member 2; tumor necrosis factor; tumor necrosis factor (TNF superfamily, member 2); tumor necrosis factor alpha; Tumor necrosis factor ligand superfamily member 2; tumor necrosis factor-alpha |
| 产品简介 (Background) |
Beyotime's Recombinant Human TNF-α contains soluble cytokines (77-233aa) of the protein. This protein was expressed in E.coli and purified without tag. It is expressed as soluble trimer, which has been validated by Size-exclusion chromatography. Apoptosis induced experiments confirmed that the protein had high biological activity. TNF-α is a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily，and it produced by various immune cells, including T cells, B cells, natural killer cells and macrophages. As a transmembrane protein, TNF-α's membrane-bound site can be cleaved by the metalloprotease ADAM17/TACE, releasing the soluble cytokine into the extracellular space to form polymers [1-2]. The cellular response to TNF-α is mediated through the receptors TNFR1 and TNFR2. TNFR1 can be activated by both the membrane-bound and soluble trimeric forms of TNF-α, and resulting the downstream apoptosis signaling pathway. While the TNFR2 responds to the membrane-bound form of TNF-α, and promotes T cell activation and proliferation [3]. TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection. The role of TNF-α in autoimmunity is underscored by blocking TNF-α action to treat rheumatoid arthritis and Crohn’s disease. |
| 产品用途 (Applications) |
Sandwich ELISA, Cell Culture, Functional Studies, Mass Spectrometry, HPLC, SDS-PAGE |
| 外观 (Physical appearance) |
Liquid |
| 活性 (Biological activity) |
Fully biologically active when compared to standard. The ED50 as determined by the dose-dependent Killing/apoptosis of L-929 cells is 0.05ng/ml corresponding to a Specific Activity of 2×107 units/mg. |
| 浓度 (Concentration) |
1mg/ml |
| 纯度(Purity) | Sterile, >98% as determined by SDS-PAGE |
| 储存液 (Storage buffer) |
PBS (pH7.4) |
本产品纯度高，并经过滤除菌处理。本产品经SDS-PAGE电泳检测蛋白纯度和分子量参考图1。本产品经过滤除菌处理，可以直接用于培养细胞的刺激。
图1. 碧云天Recombinant Human Soluble TNF-α (Trimeric form) (P5320)的SDS-PAGE电泳检测图。本产品经BeyoGel™ Plus PAGE预制胶(Hepes, 4-20%, 10孔) (P0523)电泳，蛋白Marker (M)为BeyoColor™彩色预染蛋白分子量标准(6.5-270kD) (P0071/P0072)，后续经BeyoBlue™考马斯亮蓝超快染色液(P0017F)染色。实际效果会因样品种类、检测仪器等的不同而存在差异，图中数据仅供参考。
本产品可有效激活NF-κB的核转运信号通路，使p65 (REL-associated protein, Rel-A)转移到细胞核内。稳定表达EGFP-p65的HeLa细胞(C8001)经终浓度为10ng/ml的本产品刺激后EGFP-p65进入细胞核，需要注意的是随着时间的延长EGFP-p65会出核再次回到细胞质中，因此加入本产品后观察EGFP-p65进入细胞核理想的时间为15-60分钟(图2)。
图2. 碧云天Recombinant Human Soluble TNF-α (Trimeric form) (P5320)激活NF-κB信号通路诱导核转运的效果图。稳定表达EGFP-p65的HeLa细胞(C8001)加入或不加入终浓度为10ng/ml的本产品，分别在0、30、60和120分钟用荧光显微镜进行拍照。可见在静息状态(Inactive state)下(Control)，EGFP-p65均匀分布在细胞质中(第1行)；加入TNF-α刺激后，激活状态(Active state)下EGFP-p65进入细胞核内(第2行，30分钟)，但是随着时间的延长EGFP-p65会出核再次回到细胞质中(第2行，120分钟)。实际效果会因实验仪器、检测条件等的不同而存在差异，图中效果仅供参考。
本产品与SM-164 (XIAP抑制剂)联合使用可有效诱导细胞凋亡。100nM SM-164 (XIAP抑制剂) (SC0114)与不同浓度的本产品组成的细胞凋亡诱导剂，诱导BGC-823 (人胃癌细胞)(C6123)细胞凋亡的效果参考图3。
图3. 碧云天Recombinant Human Soluble TNF-α (Trimeric form) (P5320)诱导细胞凋亡效果图。BGC-823 (人胃癌细胞) (C6123)经100nM SM-164 (XIAP抑制剂) (SC0114)与相应浓度的本产品组成的细胞凋亡诱导剂处理5-6小时，后续采用YO-PRO-1 (凋亡与坏死细胞绿色荧光探针) (C2022)对细胞进行染色。图中可见TNF-α三聚体(Trimer)的本产品比常规的TNF-α单体(monomer)有更强的细胞凋亡诱导效果。实际效果会因实验仪器、检测条件等的不同而存在差异，图中效果仅供参考。
本产品经尺寸排阻层析(Size-exclusion chromatography, SEC)验证为三聚体。人体中TNF-α为26kDa的单次跨膜蛋白，TNF-α被TACE/ADAM17 (TNF-α Converting Enzyme/A Disintegrin and Metalloprotease 17)在Ala76-Val77之间切割，成为游离于细胞膜外可溶形式的TNF-α (77-233aa)，无论是单次跨膜的TNF-α，还是游离于细胞膜外可溶形式的TNF-α，都以同源三聚体的构象行使其生物活性[4]。参考图4，本产品经16/60 Beyodex™ 200pg验证为三聚体。
图4. 碧云天Recombinant Human Soluble TNF-α (Trimeric form) (P5320)经使用16/60 Beyodex™ 200pg进行凝胶过滤层析(也称尺寸排阻层析)分析确认为三聚体的参考图。牛血清白蛋白(Bovine Serum Albumin, BSA)分子量约为66kDa，是一种常见的蛋白分子量标准，在使用16/60 Beyodex™ 200pg检测中A280nm实际出峰位置为78min (见绿色实线)。如果TNF-α为单体形式(TNF-α Monomer, 17.3kDa)，那么在使用16/60 Beyodex™ 200pg检测中A280nm理论出峰位置应为95min (见紫色虚线)。本产品在使用16/60 Beyodex™ 200pg检测中A280nm实际出峰位置为86min (见蓝色实线)，符合TNF-α三聚体形式(TNF-α Trimer, 51.9kDa)的出峰位置。实际检测结果可能会因样品和检测条件等的不同而存在差异，结果图仅供参考。
包装清单：| 产品编号 | 产品名称 | 包装 |
| P5320-10μg | Recombinant Human Soluble TNF-α (Trimeric form) | 10μg |
| P5320-100μg | Recombinant Human Soluble TNF-α (Trimeric form) | 100μg |
| P5320-1mg | Recombinant Human Soluble TNF-α (Trimeric form) | 1mg |
| — | 说明书 | 1份 |
-20℃保存，一年有效。-80℃保存，可以保存更长时间。
注意事项：由于蛋白的每次冻融均会引起部分失活，所以首次配制成相应浓度的储存液后，须分装后-20℃或更低温度冻存，尽量避免反复冻融。
本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
