Rabilloud T, Vincon M, Garin J
CEA, Laboratoire de Biologie Moléculaire du Cycle Cellulaire, INSERM U309, DBMS/BMCC, CEN-G, Grenoble, France.
Electrophoresis. 1995 Aug;16(8):1414-22. doi: 10.1002/elps.11501601234.
To improve the efficiency of one- and two-dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N,N,N',N'-tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two-dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N-terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.
为提高用于微量制备目的的一维和二维电泳效率，人们研究了使用除标准的N，N，N'，N'-四甲基乙二胺（TEMED）/过硫酸盐体系以外的其他引发剂聚合的凝胶用于十二烷基硫酸钠电泳的情况。我们在此表明，最近描述的由亚甲蓝、甲苯亚磺酸盐和二苯基碘鎓氯化物组成的光引发剂体系会导致分辨率降低。如果用核黄素代替亚甲蓝，分辨率可以恢复。这些体系也已用于评估毫克级蛋白质上样量的二维电泳。与聚合体系无关，棒状凝胶用于第一维的分辨率较低，凝胶条带的分辨率会增加，而使用固定化pH梯度时分辨率会进一步提高。同样在此，只有核黄素/亚磺酸盐/碘鎓体系产生的分辨率与标准TEMED/过硫酸盐体系获得的分辨率相当。用核黄素/亚磺酸盐/碘鎓体系聚合的凝胶在印迹后进行N端微量测序时比用标准TEMED/过硫酸盐体系聚合的凝胶产生更好的结果。
