Protocol

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration

This protocol was adapted from "Preparative 2D Gel Electrophoresis with Immobilized pH Gradients," Chapter 4, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

The equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.