Two‐dimensional electrophoresis with immobilized pH gradients in the first dimension: Protein focusing as a function of time

Since the introduction of immobilized pH gradient (IPG) electrophoresis, optimization of sample entry has been a major concern. Partial sample entry, associated with smearing of proteins, has led investigators to propose the use of hybrid IPG‐carrier ampholyte focusing for adequate protein separation. We have examined the effects of adding carrier ampholytes to the protein sample, preelectrophoresis and the site of sample application on sample entry, resolution and pattern stability for IPG gels, used as a part of two‐dimensional electrophoresis. The addition of carrier ampholytes to the sample increased the rate of entry of proteins into the gel while preelectrophoresis had an opposite effect. There was a substantial difference in protein entry between sample application at the anode vs. cathode. After an adequate focusing time the final focusing pattern was remarkably similar and consisted of several hundred well‐resolved polypeptides, irrespective of the initial starting conditions. Therefore IPG electrophoresis, without addition of carrier ampholytes to the gel, results in the high resolution separation of complex protein mixtures.