2021 Volume 65 Issue 1 Pages 17-21
Western blotting (WB) and Enzyme-linked immunosorbent assay (ELISA) are major immunological methods for protein analysis. However, it has been thought that general WB is not appropriate to analyze small proteins such as peptide hormones because of difficulty of retention of peptides on a blotting membrane. We aimed to resolve the issues by using diabetes-associated peptide hormones such as insulin as target proteins. We proved that insulin was washed-out from a PVDF membrane during procedures after electro-blotting and demonstrated that addition of “fixation step” in WB was effective for retention of proteins on the blotted PVDF membrane. Also, the modified WB was applicable to other diabetes-associated peptide hormones. These findings mean that the modified WB can contribute to qualitative or quantitative analyses of various peptides and proteins by providing analytical information based on electrophoresis. Furthermore, this paper introduces several technical tips of the improved WB.