肺组织蛋白质组双向电泳技术的建立

ESTABLISHMENT OF TWO-DIMENSIONAL GEL ELECTROPHORESIS FOR PROTEOMES OF RAT LUNG TISSUE

目的对肺组织蛋白质组分析中的双向电泳 (2 -DE)技术进行质量控制。方法通过多种条件的组合与优化 ,建立以物理和化学两种方法充分裂解组织细胞 ,并采用低温操作、加入蛋白酶抑制剂等防止蛋白降解 ,最后离心去除不溶性杂质的肺组织蛋白质组 2 -DE样品制备方法。第 1向电泳为固相pH梯度等电聚焦、第 2向电泳为垂直平板SDS -PAGE(T =13)。结果通过对蛋白定量、样品制备、第 1向和第 2向电泳中的各个实验环节进行控制和优化 ,正常大鼠肺组织在 18cmpH 3- 10L的胶上可分离 72 0个点左右 ,蛋白点平均匹配率为 72 .2 %。结论建立了肺组织蛋白质组双向电泳技术 。

Objective To establish the technique of two-dimensional gel electrophoresis(2-DE) for proteomic analysis of rat lung tissue. Methods By combination and optimization of various conditions, a sample preparation method was established The lung cells were sufficiently lysed by using physical and chemical techniques. Protein degradation was minimized by operation at lower temperature, and adding protease inhibitor. Then the insoluble impurities were removed by centrifugation. The first-dimensional electrophoresis was immobilized pH gradients isoelectric focusing and second-dimensional electrophoresis was vertical electrophoresis of SDS-PAGE(T=13%). Results By control and optimizition of many experimental conditions, the tehnique could detet about 720 spots on 18 cm IPG strip(pH3-10 L)for mormal rat lung tissue, and the match rate of protein spots was 72.2% Conclusion The technique of 2-DE which has been established is the basis of work later on.