DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 7

PAGE gels can be formed in tubes or as vertical “slab” gels, which
are formed and run between two glass plates. PAGE gels are re-
ferred to by the percentage of acrylamide in the gels (4% PAGE gel,
10% PAGE gel, etc.). The percent acrylamide is varied depending on
the nature and size of the macromolecule being separated and the
method of separation used. In general, lower percentage gels are used
for resolving nucleic acids or for isoelectric focusing, in which pro-
teins are separated based on charge characteristics. Higher percent-
age gels (10% or greater) are normally used for separating proteins in
SDS-PAGE.
SDS-PAGE
SDS (sodium dodecyl sulfate, also known as sodium lauryl sulfate)-
PAGE is one of the most widely used methods for analyzing proteins. It
is quantitative and separates proteins according to their size. Therefore,
SDS-PAGE is widely used to determine the molecular mass of proteins
(although it is inaccurate in some cases), to monitor protein purity, and,
in conjunction with western blot analysis, to identify the presence of
proteins in complex mixtures, to name but a few of the many uses of
this technique. The development of the discontinuous buffer systems
by Laemmli in 1970 (5) that are still used today greatly improved the
resolution and reproducibility of SDS gels, and is generally credited with
its popularity.
The power of the SDS-PAGE system relies not only on the gel but
on the denaturing property of SDS. SDS is a negatively charged (an-
ionic) detergent (you find it in some soaps and shampoos). When pro-
tein mixtures are boiled in SDS-containing buffers, they denature (unfold
and assume a more linear configuration). The SDS binds to the dena-
tured proteins quantitatively: the number of SDS molecules bound is
proportional to the number of amino acids in the protein. The proteins
thus have an overall negative charge, irrespective of their native charge.
When proteins are denatured in SDS and subjected to PAGE, their mo-
bility is inversely proportional to the log of their molecular weight, a
property first reported in 1965 (6). Thus, large proteins migrate slower,
whereas the smaller proteins migrate more quickly and move further
into the gel.
Most SDS-PAGE gels are prepared with a narrow “stacking gel”
above the primary separating gel. The stacking gel is a low percentage
polyacrylamide; when the proteins migrate through the stacking gel and
hit the resolving gel, it creates what has been referred to as a “traffic
jam”, such that all the proteins enter the resolving gel at essentially the
same time, improving resolution and reproducibility.