Analysis of protein complexes in mouse ES cells

小鼠 ES 细胞中蛋白质复合物的分析

• 批准号: 7732290
• 负责人: Minoru Ko
• 金额: $ 32.01万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732290
• 关键词: Aging; Animal Model; Antibodies; Cells; Complex; Condition; Databases; ES Cell Line; Embryo; Gel; Goals; Hour; Laboratories; Maintenance; Mouse Protein; Mus; Open Reading Frames; Organ; Procedures; Protein Analysis; Protein Overexpression; Proteins; Replacement Therapy; Silver Staining; Spectrophotometry; Stem cells; Tetracycline; Tetracyclines; Work; cell bank; embryonic stem cell; pluripotency; protein protein interaction; scale up; self-renewal; transcription factor

During the last year, we have optimized the conditions to purify protein complexes using antibody against FLAG-tag from mouse ES cells. We used mouse ES cells, where a FLAG-tagged transcription factor can be overexpressed in the absence of tetracycline for 72 hours. The purified protein complex was first analyzed by silver-stained gels and subsequently analyzed by mass spectrophotometry. The results indicate that our procedure works well. We are currently scaling up the procedure to analyze protein complexes for the initial 60 TFs. To aid in the protein complex analysis, we have also developed a database of mouse protein-protein interactions by transferring the protein-protein interaction information experimentally obtained from other model organisms.

去年，我们优化了使用针对小鼠 ES 细胞中 FLAG 标签的抗体纯化蛋白质复合物的条件。我们使用小鼠 ES 细胞，其中带有 FLAG 标签的转录因子可以在没有四环素的情况下过表达 72 小时。纯化的蛋白质复合物首先通过银染凝胶进行分析，随后通过质谱进行分析。结果表明我们的程序运行良好。我们目前正在扩大分析最初 60 个 TF 的蛋白质复合物的程序。为了帮助蛋白质复合物分析，我们还通过转移从其他模型生物体实验获得的蛋白质-蛋白质相互作用信息，开发了小鼠蛋白质-蛋白质相互作用的数据库。