FACTORS AFFECTING POLYACRYLAMIDE-GEL ELECTROPHORESIS AND ELECTROBLOTTING OF HIGH-MOLECULAR-WEIGHT MYOFIBRILLAR PROTEINS
FACTORS AFFECTING POLYACRYLAMIDE-GEL ELECTROPHORESIS AND ELECTROBLOTTING OF HIGH-MOLECULAR-WEIGHT MYOFIBRILLAR PROTEINS
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DOI：
10.1016/0003-2697(89)90116-4
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发表时间：
1989-08-01
影响因子：
2.9
通讯作者：
GREASER, ML
中科院分区：
文献类型：
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作者：
FRITZ, JD;SWARTZ, DR;GREASER, ML
使用常规程序很难对肌肉肌原纤维的高分子质量蛋白质（> 500 kDa）进行电泳。这些蛋白质的迁移率受到样品缓冲液中的加热时间、上层储存缓冲液中 2-巯基乙醇的使用以及堆积十二烷基硫酸钠凝胶系统中分离凝胶的 pH 值的影响。将样品加热 4 分钟（相对于更短的时间），向上层储液缓冲液中添加 2-巯基乙醇，并将分离凝胶的 pH 值降低至 8.6，所有这些都增强了聚丙烯酰胺凝胶上高分子量蛋白质的迁移率和分离度。发现蛋白质样品缓冲液中常用的巯基还原剂（2-巯基乙醇和二硫苏糖醇）会在电泳染料前沿迁移。在上层储存缓冲液中加入 10 mM 2-巯基乙醇或用 N-乙基马来酰亚胺封闭游离的巯基可防止电泳过程中分子间二硫键的形成。在用于电印迹的缓冲液中添加 10 mM 2-巯基乙醇也提高了蛋白质转移至硝酸纤维素的效率。
Electrophoresis of the high-molecular-mass proteins (> 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.