A western blotting procedure for detection of metallothionein

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.