| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| P746056-2×1μg |
2×1μg |
期货  |
| |
| P746056-2×100μg |
2×100μg |
期货 |
|
| 稳定性与储存 | -20℃保存。 |
|---|---|
| 英文名称 | Positive Control Plasmid Pair for Flag&Myc Co-IP |
| 储存温度 | -20°C储存 |
| 运输条件 | 超低温冰袋运输 |
| 产品介绍 |
阿拉丁自行研发生产的Flag&Myc Co-IP阳性对照质粒对(Positive Control Plasmids Pair for Flag&Myc Co-IP)包含pCMV-3X Flag-p53和pCMV-Myc-LTA两种质粒，可以用于在哺乳动物细胞中表达N端带有3X Flag标签的人源p53和Myc标签的LTA (large T antigen)融合蛋白。因为p53和LTA蛋白间具有较强的相互作用，本质粒对可作为共转染细胞免疫共沉淀(Co-Immunoprecipitation, Co-IP)的阳性对照使用，也可以单独转染细胞作为免疫沉淀(Immunoprecipitation, IP)的阳性对照使用。免疫沉淀或免疫共沉淀是研究蛋白或蛋白与蛋白相互作用(Protein-Protein Interactions, PPIs)的常用实验技术，通过使用特异性抗体和可结合抗体的介质(如Protein A/G Agarose或Protein A/G磁珠)，或直接使用偶联特异性抗体的介质(如琼脂糖凝胶或磁珠)，然后通过离心或磁力从溶液中分离出抗原和抗体复合物，从而将目标蛋白质从复杂样品中分离出来，随后可以用于Western印迹检测或质谱分析等1-2]。pCMV-3X Flag-p53质粒表达p53。p53中文名为细胞肿瘤抗原p53 (Cellular tumor antigen p53)，全长393氨基酸，理论分子量为43.7kDa，实际分子量约为53kDa，是调控细胞周期的重要转录因子，主要功能包括调节细胞周期、促进细胞凋亡、维持基因组稳定性和抑制肿瘤血管生成等，野生型的p53是一个重要的抑癌基因3]。pCMV-Myc-LTA质粒表达LTA。LTA即SV40大T抗原(SV40 large T antigen)，也称SV40gp6 large T antigen (Macaca mulatta polyomavirus 1)，全长708氨基酸，分子量为81.6kDa，是一种六聚体蛋白。当LTA单独表达时能诱导静止细胞的增殖和转化，可以诱导实验动物肿瘤的形成，为研究DNA复制和细胞转化提供了非常有用的模型4]。本质粒对均含有CMV启动子，可以高效启动目的蛋白在细胞中的表达，均带有氨苄青霉素(Ampicillin)抗性和新霉素(Neomycin)抗性。本质粒对用于磁珠法免疫共沉淀的效果参考图1A，用于琼脂糖凝胶法免疫共沉淀的效果参考图1B。图1. 阿拉丁的Flag&Myc Co-IP阳性对照质粒对用于免疫共沉淀的效果图。图A使用了Flag标签蛋白免疫沉淀试剂盒(磁珠法) 和Myc标签蛋白免疫沉淀试剂盒(磁珠法) ，图B使用了Flag标签蛋白免疫沉淀试剂盒(琼脂糖凝胶法) 和Myc标签蛋白免疫沉淀试剂盒(琼脂糖凝胶法) 。293T细胞(人胚肾细胞)单独或共转染pCMV-3X Flag-p53和pCMV-Myc-LTA质粒36小时后，使用Western及IP细胞裂解液裂解，并使用相应的标签蛋白免疫沉淀试剂盒进行免疫沉淀。泳道1为小鼠IgG磁珠或琼脂糖凝胶免疫沉淀后洗脱的样品，为免疫共沉淀的阴性对照；泳道2、3和4为磁珠或琼脂糖凝胶免疫沉淀后洗脱的样品，其中图A是经3X Flag Peptide或c-Myc Peptide洗脱后的样品，图B是经SDS-PAGE Sample Loading Buffer洗脱后的样品，从泳道4可观察到共转染的Flag-p53与Myc-LTA可以相互作用。Input即全细胞裂解液(Total cell lysate)。Western印迹成像由Imager 600化学发光成像系统完成。实际结果会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。推荐使用的测序引物序列如下：CMV-F primer: 5' -CGCAAATGGGCGGTAGGCGTG-3'BGH-R primer: 5'-TAGAAGGCACAGTCGAGG-3'pCMV-3X Flag-p53和pCMV-Myc-LTA的全序列信息:D3041-1 pCMV-3X Flag-p53-全长序列.txt；D3041-2 pCMV-Myc-LTA-全序列.txt。 注意事项： 本质粒未经阿拉丁书面许可不得用于任何商业用途，也不得移交给订货人所在实验室外的任何个人或单位。本质粒对仅经过测序、免疫沉淀和免疫共沉淀验证，未经过功能性验证。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。使用说明： 1.首次使用1μg包装的本产品时，请先取少量本质粒转化大肠杆菌，进行质粒小量、中量或大量抽提后再用于后续用途。抽提获得的质粒可以通过酶切电泳进行鉴定，或通过测序进行鉴定。 2.100µg包装的本产品质粒浓度为0.25µg/µl，共400µl。可以直接用于转染细胞。 参考文献： 1.Lee C. Methods Mol Biol. 2007. 362:401-406. 2.Gevaert K, Vandekerckhove J. Electrophoresis. 2000. 21:1145-1154. 3.Levine AJ. Cell. 1997. 88(3):323-331. 4.Xiao CY, Jans P, Jans DA. FEBS Lett. 1998. 440(3):297-301.Aladdin's Positive Control Plasmid Pair for Flag&Myc Co-IP contains two plasmids, pCMV-3X Flag-p53 and pCMV-Myc-LTA, which can be used to express human p53 and LTA (large T antigen) proteins fused with 3X Flag tag and Myc tag, respectively, at the N-terminus in mammalian cells. As p53 interacts strongly with LTA, this plasmid pair can be used as a positive control for Co- immunoprecipitation (Co-IP) of cotransfected cells or immunoprecipitation (IP) of separately transfected cells.IP and Co-IP are common experimental techniques to study protein or protein-protein interactions (PPIs) by using specific antibodies and resins capable of binding antibodies (e.g., Protein A/G Agarose or Protein A/G magnetic beads), or by employing directly the resins coupled with specific antibodies. The antigen and antibody complexes can be separated from the complicated protein solution by centrifugation or magnetic force, and then be analyzed by Western blot or mass spectrometry [1-2].The pCMV-3X Flag-p53 plasmid is designed for the expression of p53. p53 (cellular tumor antigen p53) is 393 amino acids long and has a theoretical mass of 43.7 kDa. Its main functions include cell cycle regulation, promoting apoptosis, maintaining genomic stability and inhibiting tumor angiogenesis. p53 in wild type is an important oncogene [3].The pCMV-Myc-LTA plasmid is designed for the expression of LTA. LTA (SV40 large T antigen), also known as SV40gp6 large T antigen, is a hexameric protein consisting of 708 amino acids with a molecular weight of 81.6 kDa. When expressed alone, LTA induces the proliferation and transformation of quiescent cells and can induce tumor formation in experimental animals, providing a very useful model for studying DNA replication and cell transformation [4].Both plasmids contain CMV promoter which can efficiently initiate the expression of target proteins in cells, and both carry Ampicillin resistance and Neomycin resistance genes.Please refer to Figure 1A for the Co-IP of Flag-p53 and Myc-LTA from cells co-transfected with Aladdin's Positive Control Plasmid Pair for Flag&Myc Co-IP by magnetic beads, and Figure 1B for the Co-IP of Flag-p53 and Myc-LTA by agarose gel.Figure 1. Co-Immunoprecipitation of Flag-p53 and Myc-LTA from cells co-transfected with Aladdin's Positive Control Plasmid Pair for Flag&Myc Co-IP . A. Flag-tag Protein IP Assay Kit with Magnetic Beads and Myc-tag Protein IP Assay Kit with Magnetic Beads were used for Co-IP; B. Flag-tag Protein IP Assay Kit with Agarose Gel and Myc-tag Protein IP Assay Kit with Agarose Gel were used for Co-IP. 293T cells (human embryonic kidney cells) were transfected with pCMV-3X Flag-p53 and pCMV-Myc-LTA plasmids, respectively, or co-transfected for 36 hours, lysed using Aladdin's Western and IP Cell Lysis Buffer , and immunoprecipitated. Western blot imaging was performed by Imager 600 Chemiluminescent Imaging System . This figure is for reference only, which may vary due to different experimental conditions.The recommended sequencing primers are as follows Precautions： This vector shall not be used for any commercial purposes without written permission from , and shall not be handed over to any individual or unit outside the laboratory where the order is placed.This plasmid pair has been validated by sequencing, immunoprecipitation and co-immunoprecipitation, but not validated by functional studies.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use： 1. If the D3041S package is ordered, please perform plasmid trasnformation in E. coli to amplify the plasmid prior to downstream applications. The extracted plasmids can be confirmed by enzyme digestion or DNA sequencing. 2. The D3041M package contains 100µg plasmids at a concentration of 0.25µg/µl, which can be used directly for cell transfection or enzyme digestion. |
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