Immobilized buffers for isoelectric focusing: from gradient gels to membranes.

Some major improvements to a pH gradient simulator for isoelectric focusing in immobilized buffers are here described. They allow creation of graphs (giving the profiles of pH gradient, deviation from ideal shape, buffering power and ionic strength) with up to 200 increments (which, in a 1 pH unit span means mapping of pH intervals at barely 0.005 pH increments per step). In addition, for preparative purification protocols, utilizing multicompartment electrolyzers with isoelectric, buffering membranes, this modified program allows easy calculation of the precise pI value at any point along the pH gradient used for the analytical assessment of the pI values of the proteins to be purified. At the desired pH value, reached by moving the cursor along the pH gradient, a simple clicking of the mouse button allows the display of the instant Immobiline composition responsible for that pH value. The molarity ratio of the various Immobilines present at this precise point of the pH scale in fact gives the composition (with the accompanying value of buffering power and ionic strength) of the membrane to be utilized as a pH-stat in the preparative scale run. This is the only method that allows reproducible and valid scaling up from the analytical to the preparative scale.