Effective Solubilization Procedure for Analysis of Silkworm Hemolymph Proteins by Two-Dimensional Gel Electrophoresis

Abstract

The outstanding capability of two-dimensional gel electrophoresis in separating all types of proteins basically depends on the efficiency of sample preparation. Sample preparation is one of the most critical steps in two-dimensional gel electrophoresis. Unfortunately, due to severe solubility, resolution of protein on gel is usually hampered, and thus, analysis remains a difficult task. However, technically several problems are generally encountered during protein extraction and isoelectric focusing. In the present investigation, we emphasized on evaluation and comparison of six different protein solubilization methods intended for resolving and analyzing silkworm hemolymph proteins by two-dimensional gel electrophoresis. Our findings revealed that the buffer composition of 8M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40mM Tris base, 65mM dithiothreitol, and 0.2% ampholyte can effectively solubilize and yields maximum protein spots.