Prospects and challenges of native protein separation and analysis using non-denaturing two-dimensional gel electrophoresis

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  • 非変性2次元ゲル電気泳動法によるネイティブタンパク質の分離分析法の展望と課題

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<p>The separation of native proteins in cytosol of mouse liver using microscale non-denaturing two-dimensional electrophoresis (2DE) requires optimization according to the charges and molecular sizes of the target proteins by adjusting the mixture of ampholytes and the density of acrylamide. Isoelectric focusing (IEF) separation of sorbitol dehydrogenase, lactate dehydrogenase and malate dehydrogenase was accomplished when 5.0% pharmalyte pH 3–10 and 1.3% pharmalyte pH 5–8 were mixed. Additionally, the malate dehydrogenase and carboxylesterase were separated using the 2DE by successively examining their activities after the size-separation of proteins using a 6.6% acrylamide gel. Furthermore, a complex of transferrin and carboxylesterase was separated at the same position on the 2DE after a mixture of transferrin and cytosol proteins in mouse liver separated by the 2DE was detected by reversible staining with imidazole-zinc following carboxylesterase activity staining. This 2DE separation is a first step in the analysis of protein-protein interactions.</p>

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