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.

在过去的一年中，我们优化了使用针对flag标签的抗体从小鼠胚胎干细胞中纯化蛋白复合物的条件。我们使用小鼠胚胎干细胞，其中flag标记的转录因子可以在没有四环素的情况下过表达72小时。纯化后的蛋白复合物首先用银染色凝胶分析，然后用质分光光度法分析。结果表明，该方法效果良好。我们目前正在扩大这一程序，以分析最初60个tf的蛋白质复合物。为了帮助蛋白质复合物分析，我们还通过转移从其他模式生物实验中获得的蛋白质-蛋白质相互作用信息，开发了一个小鼠蛋白质-蛋白质相互作用数据库。