Similar, ampholite molecule with minimum isoelectric point will have maximum negative charge (at pH 8.8) and move fastest and reach closest towards positive electrode. The ampholite with second minimum isoelectric point will not move further because the space taken by ampholite with lower isolectric point. As ampholite has high buffering capacity at isoelectric point a lower pH is maintained. As soon as it moves forward, it will experience a pH lower than its isoelectric point. Ampholite will get positive charge and move back. This way a isoelectric point gradient is generated in solid support (like polyacrylamide). As ampholytes have high buffering capacity at isolectric point, the isoelectric point gradient effectively makes a pH gradient. Now refer to our discussion earlier in this lecture about protein in pH gradient. What happens if we use pH gradient generated by ampholytes? Protein will get separated according to isolectric point. As the pH gradient is in solid support, diffusion is minimized after electric field is stopped. Gel is sliced in small pieces and proteins with different isoelectic points are separated (Fig. 4).

Figure 4: Separation of proteins in pH gradient