Analysis of the enzymatic degradation of lysozyme fibrils using a combination method of non-denaturing gel electrophoresis and double staining with Coomassie Brilliant Blue G-250 and R-250 dyes.

Abstract

The Amyloid fibrils of proteins are involved in various diseases, such as Alzheimer's disease. To suppress such amyloid fibrils, it is essential to develop methods to elucidate their enzymatic degradation process. Lysozyme in egg white has been well studied as a model protein of amyloid fibrils. Here, we establish a method for separating and evaluating both lysozyme fibrils and their enzymatic degradation products by combining non-denaturing gel electrophoresis and anionic dye staining with Congo red and two Coomassie brilliant blue (CBB) dyes. By combining non-denaturing gel electrophoresis and amyloid-specific Congo red staining, the separation site of lysozyme fibril was stained explicitly by Congo red and identified on the gel, and the amount of lysozyme fibrils decreased following the enzymatic degradation of lysozyme fibrils. Both lysozyme fibrils and their enzymatic degradation products were separated and examined by combining non-denaturing gel electrophoresis and double staining with CBB G-250 and R-250 dyes. Protein stained with negatively charged colloidal CBB G-250 could migrate to the anode side of electrophoresis. Following gel electrophoresis, noncolloidal CBB R-250 was used to detect lysozyme fibrils and the enzymatic degradation products. This method can be applied to investigate the enzymatic degradation process of amyloid fibrils.