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以建立的毛细管电泳(CE)-激光诱导荧光(LIF)检测蛋白质的方法对提取肺癌及癌旁正常组织蛋白质混合物(变性/活性)差异进行检测.采用异硫氰酸荧光素(FITC)为衍生剂,电泳缓冲液为1×TBE(TBE为Tris-硼酸-EDTA,变性电泳pH 10.0,活性电泳为pH 8.3且含有2 mg/L考马斯亮蓝),分离电压15 kV,柱温15℃,电动进样(10 kV×10 s),激发波长/发射波长=488/520 nm检测时,肺癌及癌旁正常组织蛋白质混合物样品得到较好分离且有明显差异.与目前常用蛋白分析方法:变性SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及活性蓝绿温和胶电泳(BN-PAGE)进行比较.BN-PAGE结果显示肺癌组织相比正常组织有较明显蛋白种类差异;SDS-PAGE结果表明一些蛋白质表达量差异也是肺癌及癌旁正常组织的显著差别,且主要集中在20～116 kDa.CE-LIF检测结果与PAGE结果大致相同,且CE-LIF检测蛋白质的灵敏度高于PAGE,能更准确反映肺癌及癌旁正常组织的蛋白质差异.结论是CE-LIF可用于蛋白质差异检测,时间短,效果较好,对活性蛋白质进行分析体现了其优点:可提供较强的动力——电压,及强动力下良好的温度稳定性.
The difference of denatured / active proteins in lung cancer tissues and adjacent normal tissues was detected by the established capillary electrophoresis (CE) - laser induced fluorescence (LIF) assay.Using fluorescein isothiocyanate (FITC) as a derivative The electrophoresis buffer was 1 × TBE (TBE was Tris-boric acid-EDTA, denaturing electrophoresis pH 10.0, pH 8.3 and 2 mg / L Coomassie brilliant blue). The separation voltage was 15 kV and the column temperature was 15 ℃. The electric (10 kV × 10 s) and excitation / emission wavelength = 488/520 nm, the protein mixtures of lung cancer and adjacent normal tissues were well separated and obviously different from the conventional methods of protein analysis: denatured SDS PAGE-PAGE and BN-PAGE.The BN-PAGE results showed that there were more obvious protein types in lung cancer than in normal tissues, and SDS-PAGE showed that Some differences in protein expression were also significant differences between lung cancer and adjacent normal tissues, and mainly concentrated in 20 ~ 116 kDa.CE-LIF test results and PAGE results are roughly the same, and CE-LIF protein detection sensitivity higher than PAGE, can be more Accurately reflect lung cancer and adjacent normal The results showed that CE-LIF could be used for protein differential detection with short time and good effect. The analysis of active protein shows its advantages: it can provide stronger motive power-voltage and good temperature under strong motive force stability.