Development of a New Type of Two-Dimensional Electrophoresis and Its Application to the Analysis of Alkaline Phosphatase Isoenzymes in Sera of Pregnant Woman

1) The author developed a highly sensitive staining method of alkaline phosphatase (ALP) by using an indigo derivative (TBIP) as the substrate together with an accelerator tetrazolium (NBT). By this system, 2x10^<-5> IU of ALP activity or ALP activity in 1 μl normal serum can be demonstrated as a distinctive violet band. This staining method can also be coupled with the CBBG-protein staining. By this double staining, each ALP isoenzymes can be easily identified in relation to the localization of some known serum proteins on the protein-map. 2) The author developed a new type of two-dimensional PAG electrophoresis (2-DE). The first electrophoresis is capillary isoelectric focusing of 6% PAG, in pH range 3.5-10. The second electrophoresis is 12.5% PAG slab electrophoresis using Cl^-/glycinate^- stacking system. The procedure of this 2-DE system is simple and sensitive enough to sharply separate ALP isoenzymes in the liver or placenta.