Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Protein Separation Methods
Experimental Methods of Electrophoresis - Isotachophoresis

In this method, charged particles are first separated according to their charge and mobility, and then migrate in an electric field at equal velocities.

During the experiment, the sample is injected between the leading and terminating electrolytes. The leading ion has a higher mobility than the sample ions, whereas the terminating ion has a lower mobility. The leading, terminating, and sample ions must all share the same counterion.

The leading ions migrate toward the anode faster than the sample particles. This establishes a gradient in the total ion concentration and, consequently, an electric field strength gradient (due to the variation in solution resistance). This causes an increase in the migration velocity of the sample ions. However, the latter cannot penetrate the leading ion zone because the electric field strength in this region is lower, causing the sample ions to slow down. As a result, a self-sharpening boundary is formed between the leading and sample ions.

For the same reason, the terminating ions cannot overtake the sample ions, since the latter essentially act as leading ions for the terminators. Furthermore, because the sample ions themselves possess differing electrophoretic mobilities, they sort themselves out in a similar fashion into leading and trailing zones. As a result, the Components of the analyzed mixture separate into distinct zones that ultimately migrate one after another at the same velocity.

If ampholytes are added to the sample, they position themselves between the individual components (zones) of the mixture, thereby ensuring a sharper Separation of the zones.

Such additives are referred to as "spacers" (separating or spacing agents). They essentially act as buffers or cushions between the individual components of the mixture.

Isotachophoresis is generally carried out in PAGE. In terms of resolving power, isotachophoresis approaches that of IEF.