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
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
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DOI：
10.1007/s44211-022-00229-w
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发表时间：
2022-11-30
影响因子：
1.6
通讯作者：
Shimazaki, Youji
中科院分区：
文献类型：
--
作者：
Nagata, Kenichiro;Ashikaga, Ryo;Shimazaki, Youji
淀粉样蛋白原纤维参与多种疾病，如阿尔茨海默病。为了抑制这种淀粉样纤维，必须开发方法来阐明其酶促降解过程。白色蛋白中的溶菌酶作为淀粉样纤维的模型蛋白已被充分研究。本文建立了一种非变性凝胶电泳和刚果红和两种考马斯亮蓝（CBB）阴离子染料染色相结合的分离和评价溶菌酶原纤维及其酶降解产物的方法。将非变性凝胶电泳和淀粉样蛋白特异性刚果红染色相结合，刚果红染色明确了溶菌酶原纤维的分离位点，并在凝胶上进行了鉴定，溶菌酶原纤维的数量随着溶菌酶原纤维的酶降解而减少。采用非变性凝胶电泳和CBB G-250和R-250双染色相结合的方法，对溶菌酶原纤维及其降解产物进行了分离和检测。带负电荷的胶体CBBG-250染色的蛋白质可以迁移到电泳的阳极侧。凝胶电泳后，非胶体CBBR-250被用来检测溶菌酶纤维和酶降解产物。该方法可用于研究淀粉样纤维的酶降解过程。
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.