Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 14)

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SDS-PAGE
3
3.1 Introduction
Mini-PROTEAN ® TGX ™ (Tris-Glycine eXtended shelf life) gels provide a versatile system for separating
proteins by either molecular weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter
4 for native PAGE applications and protocols.) This versatility is possible because the gels are made
without SDS; this allows the sample buffer and running buffer to determine the separation mechanism.
SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility
(glycinate and chloride) form a moving boundary when voltage is applied. Proteins have an
intermediate mobility that causes them to concentrate, or stack, into a narrow zone at the beginning
of electrophoresis. As that zone moves through the gel, the sieving effect of the polyacrylamide gel
matrix causes proteins of different molecular weighs to move at different rates. This stacking effect is
responsible for the high resolving power of SDS-PAGE: the sample is loaded in a relatively broad zone,
and the moving boundary concentrates the proteins into sharp bands prior to separation.
Protein samples for SDS-PAGE are prepared using SDS and a thiol reducing agent, usually
β-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlike
shape and similar mass-to-charge ratio. The reducing agent disrupts disulfide bonds between and
within proteins, allowing complete denaturation and dissociation. Heat treatment in the presence of
SDS and reducing agent effectively eliminates the effects of native charge and higher order structure on
electrophoretic mobility, so the migration distance depends primarily on molecular weight.
Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relative
mobility (R
f
) of the protein (R
f
= distance migrated by the protein/distance migrated by the dye front)
or by using the point-to-point semilog interpolation method in Quantity One ® or Image Lab ™ software.
Refer to bulletins 3133, 3144, and 10014472 for more information.
3.2 Mini-PROTEAN TGX and
Mini-PROTEAN ® TGX Stain-Free ™ Gels
Mini-PROTEAN TGX gels are Laemmli-like gels that have a proprietary modification that extends shelf
life to 12 months and enhances separation characteristics relative to conventional gel types. They are
run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer, and they generate
protein migration patterns that are similar to those observed with standard Laemmli Tris-HCl gels.
Two types of TGX formulations are available:
n
Mini-PROTEAN TGX — Laemmli-like, extended shelf life gels
n
Mini-PROTEAN TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo
compounds that allow rapid fluorescent detection of proteins with the stain-free system,
eliminating staining and destaining steps for faster results (see Chapter 5 for more details)