SDS/POLYACRYLAMIDE GEL ELECTROPHORESIS
INTRODUCTION: An Overview of the Method
SDS/Polyacrylamide Gel Electrophoresis is a method used to separate proteins by
molecular weight alone, thus allowing the researcher to isolate a specific sized protein
from a mixture of many similar molecules. Isolation of proteins is a valuable tool for
molecular biologists, for isolation allows one to clone a protein of interest for further
study, as well as to investigate how proteins interact to carry out a specific function.
Proteins are instrumental in nearly everything an organism does, including transport
(hemoglobin), storage (ovalbumin), defense (antibodies), and many more, some still
unknown to science (Campbell 1996).
To better understand how SDS/PAGE works, we must first investigate all components
of this procedure by asking what role they play and what results they yield. The
components in question are SDS and Polyacrylamide Gel Electrophoresis (PAGE).
THE COMPONENTS:
SDS (Sodium dodecyl Sulfate):
This is a detergent, or a soap, and can thus work to disrupt a protein's specific
conformation by dissolving the hydrophobic molecules on the inside of the
polypeptide's tertiary structure, as well as disrupting hydrogen bonds between the
parallel regions of the alpha helix and the pleated sheet (Campbell, 1996, White
1995). SDS carries out this task by wrapping around the polypeptide backbone
(Purves 1998.) Because proteins come in all shapes, folds and coils, it is important to
even the playing field by transforming all molecules to their primary, linear structure
(FIG. 1).
Figure 1. This is a cartoon showing a protein with hydrophobic interactions along the
inside of the polypeptide. These interactions give the protein a specific conformation
which helps determine function, but all secondary and tertiary structures must be
disrupted before the proteins are loaded into the gel. At the bottom, the same protein
is shown in its linear form after the the SDS "wrapped " around it, eliminating all
folds and coils and coating the protein with a negative charge. This image was
provided by Malcolm Campbell, 1998. Image Source
By eliminating the differences in conformation, we can now separate the proteins on
the basis of molecular weight alone as they move at different rates through the gel.
Not only does SDS transform proteins into their primary structure, but it coats each
protein with a negative charge. This ensures that proteins will migrate to the positively
charged side if the gel. In summary, SDS takes the many folds, coils and tangles that
make up protein structure and straightens each one into an identical conformation,
coating each protein with a negative charge. SDS prepares the protein for the next
step: PAGE.
PAGE (Polyacrylamide Gel Electrophoresis):
Polyacrylamide is a polymer of acrylamide monomers (Campbell 1998). When this
forms into a gel, it consists of small pores that make a labyrinth of tunnels and
channels through which molecules can migrate. Polyacrylamide, rather than agarose,
is the medium of choice for separating proteins by size because its small pore size is
necessary for retardation of small molecules (Purves 1998). If run on an agarose gel,
proteins, large and small, could migrate freely and end up at the same location.
Consider the proteins, denatured and coated with a negative charge with SDS, as each
moves through the gel. Most proteins will be of different lengths, for different
numbers of amino acids bond to form the primary structure of a protein. Proteins will
vary in their number of nucleotides, and thus will have different molecular weights. If
you load the proteins at the negative charged pole, all proteins will migrate towards
the positive pole at different rates as the larger ones find it more difficult to fit through
the smaller pores. Hitting such a roadblock forces the molecule to back up and try a
different route, slowing it down in the process. The smaller proteins can fit through
these tighter channels, and thus have more options on paths to take. Not hindered by
as many roadblocks, the smaller proteins will migrate through the gel at a faster rate.
What you should end up with is a gel that shows the smaller, faster proteins located
near the positive pole by a band, and the larger, slower proteins nearer to the negative
pole, shown by another band (Fig. 2).
Figure 2. This is photograph of an SDS/PAGE after the proteins migrated through the
labyrinth of polyacrylamide. This photograph was ptaken by Byron Faler, Tom
Beadle and Dr. John Williamson of the Davidson College Biology department on
Sept. 17, 1997. Image source
Of course, the number of different sized proteins will determine the number of bands
you see, but this procedure does not guarantee that all proteins located at a band will
be the same. Polyacrylamide separates proteins by molecular weight alone, neglecting
to differentiate between amino acid sequence. It is possible to have two proteins of the
same molecular weight with differing amino acid construction (Campbell 1998).
For links to sites about SDS/PAGE, select from the following: