Chapter 23 - High-Resolution Two-Dimensional Electrophoresis with Immobilized pH Gradients for Proteome Analysis

Abstract

Two-dimensional gel electrophoresis (2D PAGE) separates proteins according to isoelectric point (pI) in the first dimension and molecular mass (Mr) in the second dimension, by coupling isoelectric focusing (IEF), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For proteome analysis, it is essential to generate reproducible, high-resolution protein separations. The problem of limited reproducibility is largely due to the synthetic carrier ampholytes (CA) used to generate the pH gradient required for IEF, for reasons such as batch-to-batch variability of CAs, pH gradient instability over time, cathodic drift etc. with resultant loss of alkaline proteins. Prior to IEF, the dried IPG strips must be rehydrated to their original thickness of 0.5 mm. IPG DryStrips are rehydrated either with sample already dissolved in rehydration buffer or with rehydration buffer only, followed by sample application by cup loading. Sample in-gel rehydration is generally not recommended for samples containing very high molecular weight, very alkaline, and/or very hydrophobic proteins, as these are taken up into the gel with difficulties only.