Influence of running buffer on agarose native gel electrophoresis performance

Abstract

We previously demonstrated that agarose native gel electrophoresis using a 0.1 M histidine (His)/0.1 M MES buffer (pH 6.1) is effective for separating various proteins regardless of their charged states. In this study, we compared the performance of this buffer with different buffer systems: 0.1 M His/0.1 M MOPS (pH 6.6), 0.1 M imidazole/0.1 M MOPS (pH 7.2), 0.1 M imidazole/0.1 M HEPES (pH 7.4), 25 mM Tris/192 mM Glycine (pH 8.5) and 1×TAE (40 mM Tris-acetate, 1 mM EDTA) (pH 8.3). Using acidic BSA, neutral IgG, and basic lysozyme as model proteins, electrophoresis was conducted under identical conditions: 1% (w/v) agarose, horizontal electrophoresis, a constant voltage of 100 V, and the same run time. Among the tested systems, the 0.1 M His/0.1 M MES buffer (pH 6.1) demonstrated superior performance, providing balanced separation and clear detection of all three proteins. In contrast, while acidic BSA migrated successfully in all cases, IgG showed little migration and lysozyme displayed band tailing, smearing, or unexpectedly fast migration in the other buffer systems. Additionally, three IgG proteins with varying isoelectric points achieved optimal separation with the 0.1 M His/0.1 M MES buffer, further highlighting its effectiveness for diverse protein analyses.