NE-PER™ Nuclear and Cytoplasmic Extraction Reagents/NE-PER 核和胞浆蛋白提取提取试剂
货号：78833,78835
规格：15 ml,75 ml
价格：3203,8414
产品类型：蛋白提取/纯化
品牌：Thermo Fisher
| Thermo Scientific NE-PER核蛋白-胞浆蛋白抽提试剂盒提供了高效的细胞裂解和抽提方法，可在两个小时之内完成胞浆蛋白与核蛋白的分离操作。NE-PER试剂盒为用户提供了高效的核蛋白抽提方法，用户只需拥有台式微量离心机、离心管和移液器，进行简单的分步式细胞裂解及核蛋白与胞浆蛋白离心分离即可。NE-PER试剂盒能够高效地将胞浆蛋白与核蛋白溶解和分离为不同组分，交叉污染以及基因组DNA/mRNA的干扰均降到了最低。一旦经过脱盐或稀释，分离后的蛋白即可用于免疫分析和蛋白相互作用实验，如迁移率分析(EMSA)、免疫共沉淀(Co-IP)和拉下实验。分离胞核和制备核蛋白抽提物的方法很多，但大多数制备核蛋白抽提物的制备方法都很耗时，并需要机械匀浆、冻融循环、反复离心或透析，而这些都可能影响核蛋白的完整性。NE-PER 核蛋白-胞浆蛋白抽提试剂盒使用了基于试剂的分离方案，能够分步裂解细胞并从胞浆中分离出完整的细胞核，然后再从基因组DNA与mRNA中抽提核蛋白。这一温和的过程可在两小时之内完成，且使用培养细胞时仅需标准的桌台式离心机即可。此外，用户还可以从细胞培养物或组织样本中分离得到具有活性的核蛋白与胞浆蛋白。本试剂盒能够从两百万个细胞中提出200至500µg胞浆蛋白和100至200µg的核蛋白（浓度为1mg/mL）。一般情况下，胞浆蛋白与核蛋白之间的交叉污染在10%左右。本方法分离得到的主要是细胞核中的可溶蛋白，而非与染色质结合的蛋白或核膜整联蛋白。可通过调整核抽提试剂（NER）的体积来改变胞核抽提物的蛋白浓度，而不会对抽提效率造成显著影响。从细胞中专一地抽提核蛋白是许多基因调控研究的关键，然而现有的许多分离细胞核、制备核蛋白抽提物的方法都十分耗时，且需要进行机械匀浆、冻融循环、重复离心或透析等操作，对许多脆弱的核蛋白的完整性造成影响。NE-PER试剂盒能够克服这些问题，通过EMSA及其他分析技术为用户提供高质量的浓缩核蛋白抽提物。 |
| 优点： |
| - 快速—在两个小时之内获得胞核与胞浆内的可溶性蛋白组分； - 可靠—NE-PER试剂盒已经被950篇以上的发表文献引用； - 通用 –适用于培养细胞或组织（仅适用于新鲜样本）的核蛋白抽提； - 可扩展—两种规格试剂盒，满足从细胞和组织中抽提样本的应用需求； - 方便—操作简单，无需超速梯度离心； - 兼容性好—获得样本适合于多种下游应用，包括免疫印迹、凝胶迁移分析、蛋白分析、报告基因分析以及酶活性分析。 |
| 数据： |
图1Total protein profile of cytoplasmic and nuclear extracts using NE-PER ReagentsProtein was quantitated using Pierce Micro BCA Protein Assay Reagent (Part No. 23235). Values are the average of two separate isolations. 图2 Western blots of specific proteins fractionated using NE-PER into cytosolic extracts (C) and nuclear extracts (N)Two million A549 cells were lysed using Pierce Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER). Samples were normalized for protein concentration using Pierce BCA Protein Assay. 10µg of each cytosolic and nuclear extract sample was analyzed by 4-20% SDS-PAGE and Western blotted using specific antibodies diluted 1:1000 (HSP90, Ran, MEK1, MSH2, MCM2, HDAC1, DDX3, TopoIIb, NFκB) or 1:10000 (p53, GSK3β, β-catenin, GAPDH). Anti-mouse (H+L) HRP or anti-rabbit (H+L) HRP diluted 1:25,000 was used as the secondary antibody with SuperSignal West Dura Chemiluminescent Substrate for detection. 图3 Chemiluminescent EMSA of four different DNA-protein complexesDNA binding reactions were performed using 20fmol biotin-labeled DNA duplex (1Êbiotin per strand) and 2µL (6.8µg total protein) NE-PER Nuclear Extract prepared from HeLa cells. For reactions containing specific competitor DNA, a 200-fold molar excess of unlabeled specific duplex was used. 图4 Total protein profile of cytoplasmic and nuclear extracts prepared from different mouse tissuesSwiss Webster mouse tissues (40mg) were harvested, rinsed with PBS and lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit. Extracts were quantified using the Pierce 660nm Protein Assay Reagent (Part No. 22660). Values are the average of two separate isolations. 图5 Western blots of specific proteins from fractionated tissuesCytoplasmic and nuclear extract (10µg each) from different mouse tissue fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit was analyzed by 4-20% SDS-PAGE and Western blotting. Primary antibodies specific for the target proteins were diluted 1:1,000 (SP1, HDAC2 and NFκB p65, or 1:10,000 (GAPDH). Anti-Rabbit (H+L) HRP (Part No. 31460) diluted 1:25,000 was the secondary antibody and SuperSignal West Dura Chemiluminescent Substrate (Part No. 34076) was used for signal detection. |
| 相关产品： |
| ▪NE-PER™ Nuclear and Cytoplasmic Extraction Reagents（货号#78833/78835）▪Mem-PER™ Plus Membrane Protein Extraction Kit（货号#89842）▪Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (100X)（货号#78441/78443/78445/78447）▪Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100X)（货号#78442/78440/78444/78446） |
技术参数 产品优点 1. 快速—在两个小时之内获得胞核与胞浆内的可溶性蛋白组分；
2. 可靠—NE-PER试剂盒已经被950篇以上的发表文献引用；
3. 通用 –适用于培养细胞或组织（仅适用于新鲜样本）的核蛋白抽提。
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