Bio-Rad Gel Doc™ EZ System - Manual (Page 32)

Criterion Stain Free TM System | Criterion Stain Free Gels
18
SDS-PAGE
Criterion 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 page 20 for Native PAGE applications and protocols.) This
versatility is possible because Criterion 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 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 (DTT) 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).
Criterion Tris-HCl Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Cross linker 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6
Stacking gel 4% T, 2.6% C
Shelf life ~12 weeks; individual expiration date is printed on each
cassette