An improved protocol for two‐dimensional maps of serum proteins with immobilized pH gradients in the first dimension

Additional information is provided on the performance of two‐dimensional electrophoretic separations of human serum using immobilized pH gradients in the first dimension first described in Electrophoresis 1984. 5 , 209–216. Special attention is devoted to the effect of different sample manipulations, including the site of sample loading along the pH gradient, its concentration (moderate dilution has favorable effects for the quantitative and prompt migration of the proteins from the application well) and its pretreatment (different serum patterns result from incubation in various denaturing and non‐denaturing media). An interference to the migration from the first to the second dimension matrix is observed by high Immobiline concentrations (β power should then not exceed 3 mEq/liter pH). The presence of 2‐mercaptoethanol in the equilibration medium prior to the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis step is found essential for a proper peptide mapping.