Subcellular Protein Fractionation Kit for Cultured Cells / 培养细胞的亚细胞蛋白分离试剂盒

货号：78840

规格：35 mL

价格：7746

产品类型：蛋白提取/纯化

品牌：Thermo Fisher

Thermo Scientific培养细胞亚细胞蛋白分离试剂盒能够从5种不同的细胞区室中分离和富集蛋白。亚细胞蛋白可用于蛋白定位测定以及从特定细胞区室中富集蛋白。Thermo Scientific 亚细胞蛋白分离试剂盒包括一系列试剂，能在3小时内完成将细胞逐步裂解为功能胞浆蛋白、膜蛋白、可容性核蛋白、染色质结合蛋白和细胞骨架蛋白。每种亚细胞区室抽提物的组分间污染一般少于15%，该纯度可满足绝大部分蛋白定位和重分布实验研究。培养细胞亚细胞蛋白质分离试剂盒包含四种抽提缓冲液、稳定核酸酶和Thermo Scientific Halt 蛋白酶抑制剂混合物。每一个试剂盒包含足够分离50个20 µL的细胞沉淀，相当于大约2g细胞体。（对于组织样品，请使用我们的组织亚细胞蛋白质分离试剂盒）第一种试剂将组织匀浆，使膜选择性通透，从而释放可溶性胞质内容物。第二种试剂用于溶解质膜、线粒体膜和内质网- 高尔基体膜，但不溶解核膜。通过离心回收完整胞核后，第三种试剂将产生可溶性细胞核提取物。额外的细胞核提取是使用微球菌核酸酶进行提取以释放染色质结合的核蛋白。然后使用最后一种试剂提取回收的不可溶沉淀，用以分离细胞骨架蛋白。

特点：

• 简洁—在3小时以内即可完成抽提胞浆蛋白、膜蛋白、可容性核蛋白、染色质结合蛋白和细胞骨架蛋白中的功能性组分• 方便—方法简单，无需使用密度梯度超离• 完整—单个试剂盒即可获得胞浆蛋白、膜蛋白、可容性核蛋白、染色质结合蛋白和细胞骨架蛋白• 培养细胞—为哺乳动物细胞开发并优化。对于组织样品请使用组织亚细胞分离试剂盒• 兼容性强—抽提物与蛋白免疫印迹、蛋白定量分析、电泳凝胶迁移和报告基因以及酶活性检测等一系列下游应用兼容。

应用：

•从不同细胞区室抽提和富集蛋白•从同样的细胞分离胞浆蛋白、核蛋白、膜蛋白和细胞骨架蛋白•检测胞浆、核、膜和细胞骨架的细胞蛋白特征•蛋白转移定位研究

数据：

Western blots of fractionated cellular proteinsHeLa cells (2 x 10^6) were fractionated using the Subcellular Protein Fractionation Kit. Normalized portions of each extract (10µg) were analyzed by Western blotting using specific antibodies against proteins from various cellular compartments including cytoplasmic (HSP90), plasma membrane (EGFR), endoplasmic reticulum (calreticulin), nuclear soluble (SP1 and HDAC2); chromatin-bound (histone 3); and cytoskeleton (cytokeratin 18 and vimentin). The blots were probed with goat anti-rabbit (H+L) HRP or goat anti-mouse (H+L) HRP and detected with Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076). The results demonstrate clear fractionation of each protein with low cross contamination among fractions.CE: cytoplasmic extractME: membrane extractNE: nuclear extractCB: chromatin-bound extractPE: pellet extract Western blot analysis of protein translocationPanel A. A549 cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 20µg/mL TNFα for 20 minutes and fractionated using the Subcellular Protein Fractionation Kit. Each extract (10µg) was analyzed by Western blot using an anti-NFκB p65 antibody. Panel B. Serum-starved HeLa cells (2 x 10^6) were either mock-treated (-) or incubated (+) with 1µM PMA for 20 minutes and fractionated. Each extract (10µg) was analyzed by Western blot using an anti-PKCα antibody. Goat anti-rabbit or anti-mouse (H+L) HRP was used as the secondary antibody, and SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076) was used for detection. Schematic overview of the subcellular fractionation procedureCellular compartments are sequentially extracted by incubating cells with cytoplasmic extraction buffer (CEB) followed by membrane extraction buffer (MEB) and nuclear extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts chromatin-bound proteins from the cell pellet before adding the pellet extraction buffer (PEB) to solubilize cytoskeletal proteins.

技术参数

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