运用双向聚丙烯酰胺凝胶电泳和图像软件分析TPA处理NIH3T3细胞前后的蛋白质表达谱差异

目的建立及优化聚丙烯酰胺双向凝胶电泳技术,并应用图像软件分析伏波醇酯(TPA)刺激前后小鼠成纤维细胞NIH3T3系的蛋白质差异表达,为进一步鉴定与TPA生物学作用有关的蛋白质奠定基础。方法分别提取TPA处理前后的NIH3T3细胞总蛋白质,经双向电泳,硝酸银染色后用软件分析显示两者蛋白质的差异表达。结果软件分析显示TPA刺激前后的细胞蛋白质有明显的差异表达。结论高质量的双向电泳设备、熟练的电泳操作和图像处理技能是寻找稳定可信的功能蛋白质的必要条件。

Analysis of the difference in NIH3T3 cell protein expression profiles before and after TPA treat-ment using two-dimensional polyacrylamide gel electrophoresis and image analysis software

Objective To establish and optimize two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) for comparative analysis of the protein expression profiles in mouse fibroblast NIH3T3 cells before and after 12-O-tetradecanoy- lphorbol-13-acetate (TPA) treatment using image analysis software, so as to prepare for more intensive study in the identifi- cation of the proteins that mediate the biological effect of TPA. Methods The total cellular protein extracted from NIH3T3 cells with or without TPA treatment underwent 2-D PAGE and silver nitrate staining prior to the analysis of the differential protein expressions using image analysis software. Results Image analysis revealed obvious differential protein expressions of the cells in response to TPA treatment. Conclusion High-quality instruments for 2-D PAGE, skilled electrophoretic operation and efficient image processing are all essential in the reliable identification of stable functional proteins.