Bio-Rad Criterion™ TBE-Urea Precast Gels - Manual (Page 13)

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SDS-PAGE
3
3.1 Introduction
Criterion ™ precast gels provide versatile systems for the separation of 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 Criterion gels are made without SDS, allowing 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 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
weights 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 usually a thiol reducing agent such as
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 and 3144 for more information.
3.2 Criterion Gel Selection Guide for SDS-PAGE
A number of Criterion gel types are available for SDS-PAGE (Table 3.1) in both single and gradient
polyacrylamide percentages. Use the protein migration charts and tables to select the gel type that
offers optimum resolution of your sample:
n
Use single-percentage gels to separate bands of similar molecular weight. Optimum
separation occurs in the lower half of the gel, so use a percentage in which the protein
migrates to the lower half of the gel
n
Use gradient gels to separate samples containing a broad range of molecular weights.
Gradient gels allow resolution of both high and low molecular weight bands on the same
gel. Larger pore sizes at the top of the gel permit resolution of larger molecules, and smaller
pore sizes toward the bottom of the gel restrict excessive separation of small molecules