Bio-Rad Ready Gel Precast Gels for 2-D Electrophoresis - Manual (Page 12)

Section 3
SDS-PAGE
3.1 Introduction
Ready Gel Tris-HCl gels provide a versatile system for the separation of proteins by molecular weight (SDS-PAGE
conditions) or charge to mass ratio (native conditions). (See section 4 for native PAGE applications and
protocols.) This is possible because Ready Gel Tris-HCl gels are made without SDS, allowing the sample
buffer and running buffer to determine the separation mechanism. Historically, SDS-PAGE systems contained
SDS in both the gel and the running buffer. Reproducible SDS-PAGE separations are performed in gels
lacking SDS provided the sample buffer and running buffers contain sufficient SDS to saturate the proteins
during electrophoresis. The recommended concentration of SDS is 2% in sample buffer and 0.1% in running
buffers.
SDS-PAGE uses discontinuous chloride and glycine ion fronts to form moving boundaries that stack and
then separate SDS-coated polypeptides by molecular weight. Protein samples are prepared in a reducing
denaturing sample buffer containing either 2-mercaptoethanol or dithiothreitol as the reducing reagent, and
heat and SDS are used to denature the proteins. 2-Mercaptoethanol and dithiothreitol eliminate protein
secondary structure by reducing disulfide bonds. SDS minimizes charge variability among proteins, giving
them the same charge to mass ratio and forcing them into rod-like shapes. This effectively eliminates the
effects of protein conformation and native charge density on the electrophoretic migration distance. Molecular
weight determinations are obtained by plotting the logarithm of protein molecular mass vs. the relative
mobility (Rf = distance migrated by protein/distance migrated by dye front).
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