2023 Volume 67 Issue 2 Pages 71-75
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.
