Two-Dimensional Gel Electrophoresis with Immobilized pH Gradients

Isoelectric focusing (IEF) is a technique in protein research that has been used since 1975 to separate proteins based on their isoelectric point. When combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis, this procedure allows for high-resolution separation of cellular proteins for analytical purposes. Laboratories perform IEF by (a) using carrier ampholytes that migrate through a gel to create the pH gradient or (b) using immobilized pH gradients (IPG) that contain ampholytes bound covalently to a gel. Here we describe an IEF system that uses immobilized pH gradient (IPG) strips that undergo the desired current and voltage setting to separate proteins based on its charge in the first dimension followed by SDS PAGE to generate a two-dimensional map of serum proteins.