Aoki Y, Tohyama C, Suzuki K T
National Institute for Environmental Studies, Ibaraki, Japan.
J Biochem Biophys Methods. 1991 Oct-Nov;23(3):207-16. doi: 10.1016/0165-022x(91)90013-m.
A method to detect metallothionein (MT) by Western blotting technique is described. Cadmium-containing rat MT-I and -II were subjected to sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-PAGE) without prior reduction by 2-mercaptoethanol (2-ME), because prior reduction of MTs by 2-ME resulted in diffused bands. After SDS-PAGE, MTs in the gel were reduced by incubating in buffer containing 2-ME, and then electrophoretically transferred to a polyvinylidene fluoride membrane. Non-reduced MTs were not found to be immobilized on the membrane. Prior to electrophoresis the zinc, in zinc-containing MT samples, must be replaced with cadmium by addition of excess cadmium chloride. MTs immobilized on the membrane were detected by two methods, (1) binding of radioactive cadmium and (2) immunochemical staining using protein-A/colloidal gold conjugate, the detection limits of which were 0.4 and 0.06-0.16 micrograms, respectively.
本文描述了一种通过蛋白质免疫印迹技术检测金属硫蛋白（MT）的方法。含镉的大鼠MT-I和MT-II在不预先用2-巯基乙醇（2-ME）还原的情况下进行十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS-PAGE），因为用2-ME预先还原MT会导致条带扩散。SDS-PAGE后，凝胶中的MT通过在含2-ME的缓冲液中孵育进行还原，然后电泳转移至聚偏二氟乙烯膜上。未发现未还原的MT固定在膜上。在电泳前，含锌MT样品中的锌必须通过加入过量的氯化镉用镉替代。固定在膜上的MT通过两种方法检测：（1）放射性镉的结合；（2）使用蛋白A/胶体金复合物的免疫化学染色，其检测限分别为0.4微克和0.06 - 0.16微克。
