The analytical electrophoresis of proteins is carried out in polyacrylamide gels
under conditions that ensure dissociation of the proteins into their individual
polypeptide subunits and that minimize aggregation. Most commonly, the
strongly anionic detergent sodium dodecyl sulfate (SDS) is used in combination
with a reducing agent and heat to dissociate the proteins before they are loaded
on the gel. The denatured polypeptides bind 50S and become negatively charged.
Because the amount of SDS bound is almost always proportional to the molecular
weight of the polypeptide, and is independent of its sequence, 50S-polypeptide
complexes migrate through polyacrylamide gels in accordance with the size of
the polypeptide. At saturation, approximately 1.4 g of detergent is bound per
gram of polypeptide. By using markers of known molecular weight, it is therefore
possible to estimate the molecular weight of the polypeptide chains.
SDS-polyacrylamide gel electrophoresis is carried out with a discontinuous
buffer system in which the buffer in the reservoirs is of a different pH and ionic
strength from the buffer used to cast the gel.
The 50S-polypeptide complexes in
the sample that is applied to the gel are swept along by a moving boundary
created when an electric current is passed between the electrodes. After migrating
through a stacking gel of high porosity, the complexes are deposited in a very
thin zone (or stack) on the surface of the resolving gel. The ability of the
discontinuous buffer systems to concentrate all of the complexes in the sample
into a very small volume greatly increases the resolution of SDS-polyacrylamide
The sample and the stacking gel contain Tris-CI (pH 6.8), the upper and
lower buffer reservoirs contain Tris-glycine (pH 8.3), and the resolving gel
contains Tris-CI (pH 8.8). AI-components of the system contain 0.1% 50S. The
chloride ions in the sample and stacking gel form the leading edge of the
moving boundary, and the trailing edges of the moving boundary are a zone
of lower conductivity and steeper voltage gradient, which sweeps the
polypeptides from the sample. There, the higher pH of the resolving gel favors
the ionization of glycine, and the resulting glycine ions migrate through the
stacked polypeptides and travel through the resolving gel immediately behind
the chloride ions. Freed from the moving boundary, the 50S-polyacrylamide
complexes move through the resolving gel in a zone of uniform voltage and pH,
and are separated according to size by sieving.
Polyacrylamide gels are composed of chains of polymerized acrylamide
that are crosslinked by a bifunctional agent such as N, N’-Methylenebisacrylamide.
The effective range of separation of SOS-polyacrylamide gels
depends on the concentration of polyacrylamide used to cast the gel, and on
the amount of crosslinking.
Molar ratio of bis-acrylamide: acrylamide is 1:29.
Crosslinks formed from bisacrylamide add rigidity and tensile strength to
the gel and form pores through which the 50S-polypeptide complexes must
The sieving properties of the gel are determined by the size of the pores,
which is a function of the absolute concentrations of acrylamide and
bisacrylamide used to cast the gel.