Proteins are involved in every aspect of cellular life.
Proteins function
to transport materials across membranes, catalyze chemical
reactions, organize DNA, support the movement of materials within a
cell, and even drive movement of the entire cell. The technique
of sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS-
PAGE is a standard technique for the analysis of the protein
composition of a sample. The nature of the sample will vary
depending on the specific experiment.
Proteins differ in the number and type of amino acids that assemble to
form a polypeptide chain. The chemistry of the amino acid side chains
(R groups) determines protein folding and the overall charge of the
protein. SDS-PAGE works by separating proteins based on their
relative size. In order to do this, protein folding and the differences in
charge of the various proteins must be eliminated. Protein gel samples
are treated with the nonionic detergent, sodium dodecyl sulfate (SDS).
SDS coats the proteins with a net negative charge, masking any
charges normally present on the protein’s surface. The sample is also
treated to disrupt protein folding by the addition of chemical reagents
such as dithiothreitol (DTT) or beta mercaptoethanol (BME) and
heat. DTT and BME function to disrupt any disulfide bonds within the
protein. The combination of SDS, DTT/BME, and heat will denature or
unfold the protein.
The protein sample is loaded into a well or space that is formed at the
top of the polyacrylamide gel. Polyacrylamide is chemically
crosslinked to form a Jello-like matrix or gel. The density of the gel can
be varied based on the percent composition of polyacrylamide. High
percent acrylamide (dense) gels are useful for examining small
proteins while lower percent acrylamide (thin) gels are best for
examining larger proteins. Gels with an acrylamide concentration of
around 8–10% are standard for most research applications.
The power of SDS-PAGE comes from its ability to separate a mixture
of proteins into individual bands. Proteins migrate out from the well
and through the polyacrylamide matrix in response to an electrical
current. Recall that all the proteins in a sample have been treated with
SDS and so are now negatively charged. Negatively charged proteins
will migrate away from a anode and toward an cathode in an electrical
circuit. The speed with which the various proteins migrate will be
influenced by the degree of crosslinking of the acrylamide gel. Smaller
proteins will have an easier time moving through the crosslinked
matrix while larger proteins will move much more slowly. The net
effect is the separation of proteins based on size, with smaller proteins
at the bottom of a gel and larger proteins nearer the top. The size of a
protein can be estimated by comparing the distance a protein has
migrated relative to the movement of molecular weight standards.
These standards are a mix of proteins of known molecular weight (in
units of kilodaltons or kDa).
Protein gels must be stained to visualize the individual protein bands
before they can be analyzed. Various staining protocols exist;
however, one very common method is the use of a stain
called Coomassie Blue. The Coomassie dye has the advantage of
binding to proteins in a 1:1 ratio. This means that the staining intensity
of a protein band on the gel is a measure of the concentration of that
particular protein in the sample.
■
Name the amino acids that would contribute a positive charge to
the surface of a protein.
■
Which amino acids are involved in the formation of a disulfide
bond?
■
Conduct a search for images of a polyacrylamide gel and the
apparatus used to run the gel.
■
Explain why a small protein migrates faster than a larger protein
in a gel.
