ISOELECTRIC-FOCUSING OF PROTEINS IN SLAB POLYACRYLAMIDE-GEL PREPARED ON CELLOPHANE SHEET

A method of isoelectric focusing in slab polyacrylamide gel prepared on a sheet of cellophane was developed. A slab polyacrylamide gel containing 2% carrier ampholyte was prepared on a cellophane sheet placed on a plastic film. The gel prepared on the cellophane sheet is referred to as slab polyacrylamide gel with cellophane. Isoelectric focusing of proteins was performed in the usual manner using the slab polyacrylamide gel with cellophane. After focusing the gel with cellophane was removed from the plastic film, and staining and destaining were performed in the usual manner. After destaining, the gel surface was covered with another cellophane sheet, and dried with a gel dryer. The pattern of focused proteins was almost the same as that obtained in a gel without cellophane. Handling of the gel during staining, destaining and drying and storage of the gel was easy. The isoelectric point (pI) value of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) was studied using the present method with ribonuclease A-glutathione mixed disulfides as pI standards. Results were similar to the previous reports (T. Ubuka et al. (1987) Anal. Biochem. 167:284-289; T Ikeda et al. (1989) Acta Med. Okayama 43:1-7), but the procedure with the present method was easier and a more precise determination was possible because the changes in the gel size during staining and destaining were minimized.