Isotachophoresis of proteins in sucrose density gradients

The separation of proteins from human serum by isotachophoresis in sucrose density gradients, with mixtures of discrete amphoteric substances as spacers, is described. Open columns and columns with a dialysis membrane to hold the sucrose gradients were used. A simple algorithm based on the Kohlrausch function was used to calculate the amount of each spacer. The pH gradients generated in open columns were found to be in agreement with the calculations. The load was up to two gram proteins. The analysis of the fractions obtained after the separation showed a distribution of components similar to as analytical isotachophoresis. It is concluded that sucrose density gradients are suitable as supporting media for the preparative separation of proteins by isotachophoresis. The high resolution attained and the possibility of scaling-up the separation systems are major advantages of this system. In addition, the sample is easily and completely recoverable.