Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding ∼ 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion‐containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1–2 mM Tris base, or at the anode for gels rehydrated with 1–2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris‐containing gels, or at the cathode, for acetate‐containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading. Salt concentrations in excess of ∼ 0.15 M in the sample result in electroosmotic removal of water from the sample before the proteins can enter the gel, with resultant precipitation of the sample at the application site. Local pH extremes can be created at the application point, which may produce delayed sample entry or protein precipitation.