不同载体微球在p300与RORγt Co-IP实验效率的比较

Comparison of experimental efficiency of different carrier microbeads in p300 and RORγt Co-IP

目的:在免疫共沉淀(Co-IP)过程中使用Protein A/G-琼脂糖珠捕获蛋白和使用Protein A/G-磁珠捕获蛋白的比较分析。方法先通过共转染Flag-p300和Myc-RORγt质粒入人胚肾细胞系293T(HEK293T)进行过表达,裂解细胞制备蛋白,分别使用Protein A/G-琼脂糖珠捕获蛋白和使用Protein A/G-磁珠捕获蛋白进行Co-IP;再通过分离人脐血单个核细胞诱导分化人辅助性T细胞17(Th17)进行Co-IP比较分析;通过CCK8实验排除转染试剂等对细胞活性毒性。结果过表达的HEK293T细胞和诱导分化的Th17细胞中均显示腺病毒E1A结合蛋白(p300)与维甲酸相关孤儿核受体(RORγt)在Co-IP过程中使用Protein A/G-磁珠捕获大分子的p300蛋白效果好于中分子的RORγt蛋白,使用Protein A/G-琼脂糖珠捕获中分子的RORγt蛋白效果好于大分子的p300蛋白。结论在Co-IP过程中使用Protein A/G-磁珠捕获大分子蛋白效果较好,使用Protein A/G-琼脂糖珠捕获中分子蛋白效果较好。

Objective Comparative analysis of p300 and RORγt used Protein A/G-agarose beads to capture protein and Protein A/G-Magnetic beads to capture protein in the process of immunoprecipitation.Methods After co-transfection of Flag-p300 and Myc-RORγt plasmids into HEK293T cells for expression,the cells were lysed to prepare proteins,and Protein A/G-agarose beads capture proteins and Protein A/G-Magnetic beads capture antigens were used for Co-Immunoprecipitation(Co-IP),respectively.Human umbilical cord blood monocytes were isolated from human umbilical cord blood monocytes to induce differentiation of human Th17 cells by Co-IP comparative analysis,and CCK8 assay was used to exclude the cytotoxicity of transfection reagents.Results Both overexpressed HEK293T cells and induced differentiation of Th17 cells showed adenovirus E1A binding protein(p300)and retinoic acid-associated orphan nuclear receptor(RORγt)in the Co-IP process,using Protein A/G-magnetic beads to capture large the p300 protein of the molecule was better than the RORγt protein of the middle molecule.The use of Protein A/G-agarose beads to capture the RORγt protein of the molecule was better than that of the macromolecule p300 protein.Conclusion In the course of immunoprecipitation,it is better to capture macromolecular protein with protein A/G magnetic beads and protein A/G agarose beads to capture middle-molecular proteins,which is better than that by using protein A/G agarose beads in the process of immunoprecipitation.