Protocol

Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel

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

Following the separation of proteins by IEF, the second dimension is carried out by SDS-PAGE. This protocol details the method for casting single homogeneous SDS-PAGE gels. Homogeneous gels (with the same %T and %C throughout) offer the best resolution for a particular molecular-weight range and are commonly used because they are the easiest to pour reproducibly. The second-dimension gels can be conveniently prepared in three different formats (i.e., sizes): minigels, for use with 7-cm IEF first-dimension gels; standard gels, for use with 11-, 13-, and 18-cm IEF first-dimension gels; and large-format gels, for use with 18- and 24-cm IEF first-dimension gels. All of the gels use a common set of reagents, listed below, but differ slightly in the equipment required.