Simplified procedure for the preparation of immobilized pH gradient gels

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Abstract
Immobilized pH gradient (IPG) slab gels are conventionally formed by use of two‐chamber gradient mixers on pH‐neutralized Immobiline mixtures, co‐polymerization of the gradient of Immobilines with acrylamide at 50 °C, washing and drying of the gel to its original weight followed by pre‐electrophoresis before applying the sample. This tedious procedure was replaced by one using gradient formation by pump, eliminating pH neutralization of the monomer mixture, substituting polymerization at 50 °C for one at the temperature of electrophoresis (10 °C in this study) and omitting washing and drying of the gel prior to use. Carrier ampholyte (CA) containing IPG gels were formed in the same way except that CAs were added to the polymerization mixture. Pre‐electrophoresis of IPG gels, formed by either the conventional or the simplified procedure, was found to be detrimental to the IPG patterns of proteins. The simplified procedure also allowed one to conduct IPG electrophoresis in gel tubes, eliminating lateral zone diffusion.