一步法电泳分析肌联蛋白亚型和肌球蛋白重链

One-step gel electrophoresis of titin isoforms and myosin heavy chains by SDS-VAGE in tandem with SDS-PAGE

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。

Objctive In order to facilitate the analysis of the giant myofibrillar protein titin, a new gel electrophoresis system is developed to resolve giant titin isoforms and medium sized myosin heavy chain （MHC） at the same time. Methods A 16cm× 18 cm vertical gel electrophoresis system was utilized. Two different layers of gels were casted. The upper layer was 10 g/L sodium dodecyl sulfate agarose gel e lectrophoresis （SDS VAGE） responsible for resolving titin isoforms, and the lower layer was 60 g/L sodi um dodecyl sulfate polyacrylamide slab gel electrophoresis （SDS PAGE） for separating MHC. The gels were cooled to 8℃ and run for 5 hours. After electrophoresis, the silver staining protocol for VAGE gels and the Coomassie Brilliant Blue R 250 staining protocol for PAGE gels were adapted. Results Titin iso forms and MHC were resolved on SDS VAGE gel and SDS PAGE gel clearly in onestep. Conclusion Gel e lectrophoresis analysis of giant and medium sized proteins can be achieved by combining SDS VAGE and SDA PAGE, which can improve accuracy and precision of electrophoresis analyses.