Two-dimensional maps in the most extended (pH 2.5-11) immobilized pH gradient interval.

In conventional isoelectric focusing in soluble, amphoteric buffers, it has been quite difficult to produce two-dimensional (2-D) separations in pH intervals greater than pH 4-8. In general more alkaline proteins were analyzed by non-equilibrium IEF in the first dimension. Even with the advent of immobilized pH gradients (IPG), separations could be extended to pH gradients not wider than pH 3-10, due to a lack of suitable buffers. Since more acidic and more alkaline acrylamido buffers have recently been synthesized, we have been able to optimize what is believed to be the widest possible immobilized pH gradient, a pH 2.5-11 span. We report here for the first time 2-D separations of total tissue lysates in such extended pH 2.5-11 gradients. It appears that, with the IPG technique, close to 100% of all possible cell products can be displayed in a single 2-D map.