Protein desalting by isoelectric focusing in a segmented immobilized pH gradient

We have recently described an apparatus for protein purification based on a segmented Immobiline gel, having one or more liquid interlayers in between. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow chamber, and focusing the impurities in an Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. We now demonstrate that the present apparatus can be efficiently used for protein desalting. Hemoglobin A samples, containing 50 mM NaCl or 50 mM ammonium acetate, could be efficiently desalted in 2 h of recycling, after which the total salt content had decreased to less than 0.005 mM (a salt decrement of more than 10,000 fold the initial input). However, with polyprotic buffers (sulphate, citrate, phosphate, oligoamines) the desalting process was much slower, typically of the order of 20 h, possibly due to interaction of these species with the surrounding Immobiline matrix. In this last case, outside pH control (e.g. with a pH-stat) is necessary during protein purification, as, due to the faster removal of the monovalent counterion, the solution in the recycling chamber can become rather acidic or alkaline. It is demonstrated that the 2 extremities of the Immobiline segments facing the sample recycling chamber act indeed as isoelectric membranes, having a good buffering capacity, preventing the protein macroion from leaving the chamber by continuously titrating it to its isoelectric point.