Introduction to SDS- PAGE : Rice University

Introduction to SDS-PAGE
This material is accompanied by a presentation on protein structure and principles behind
denaturing samples and discontinuous gel electrophoresis.
The separation of macromolecules in an electric field is called electrophoresis. A very
common method for separating proteins by electrophoresis uses a discontinuous
polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the
proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). The most commonly used system is also called the Laemmli method after U.K.
Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study.
SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its
molecules have a net negative charge within a wide pH range. A polypeptide chain binds
amounts of SDS in proportion to its relative molecuar mass. The negative charges on SDS
destroy most of the complex structure of proteins, and are strongly attracted toward an anode
(positively-charged electrode) in an electric field.
Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules.
Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the
final separation of proteins is dependent almost entirely on the differences in relative
molecular mass of polypeptides. In a gel of uniform density the relative migration distance of
a protein (Rf, the f as a subscript) is negatively proportional to the log of its mass. If proteins
of known mass are run simultaneously with the unknowns, the relationship between Rf and
mass can be plotted, and the masses of unknown proteins estimated.
Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to
determine the relative abundance of major proteins in a sample, and to determine the
distribution of proteins among fractions. The purity of protein samples can be assessed and
the progress of a fractionation or purification procedure can be followed. Different staining
methods can be used to detect rare proteins and to learn something about their biochemical
properties. Specialized techniques such as Western blotting, two-dimensional electrophoresis,
and peptide mapping can be used to detect extremely scarce gene products, to find
similarities among them, and to detect and separate isoenzymes of proteins.

Molecular mass versus molecular weight

Molecular mass (symbol m) is expressed in Daltons (Da). One Dalton is defined as 1/12 the
mass of carbon 12. Most macromolecules are large enough to use the kiloDalton (kDa) to
describe molecular mass. Molecular weight is not the same as molecular mass. It is also
known as relative molecular mass (symbol Mr, where r is a subscript). Molecular weight is
defined as the ratio of the mass of a macromolecule to 1/12 the mass of a carbon 12 atom. It
is a dimensionless quantity.
When the literature gives a mass in Da or kDa it refers to molecular mass. It is incorrect to
express molecular weight (relative molecular mass) in Daltons. Nevertheless you will find the

term molecular weight used with Daltons or kiloDaltons in some literature, often using the
abbreviation MW for molecular weight.

Polyacrylamide gels for SDS-PAGE

Many systems for protein electrophoresis have been developed, and apparatus used for SDSPAGE varies widely. The methodology used on these pages employs the Laemmli method.
Reference to the Laemmli method in a materials and methods section eliminates the need to
describe the buffers, casting of gels, apparatus, etc. Unless the paper employs some
modification to the method, the only details of SDS-PAGE that should be reported in a
methods section are percent total acrylamide (%T) in a gel, relative percentage and type of
crosslinker (%C), and perhaps a reference to the gel dimensions. We use a "mini-gel" system,
with 3 1/4" x 4" gel cassettes.
SDS-PAGE can be conducted on pre-cast gels, saving the trouble and hazard of working with
acrylamide. The following description applies to shop-made casting and running apparatus
that are much cheaper than commercially available equipment. In addition to cost
effectiveness, an advantage of making one's own gels the first time is a deeper understanding
of the process.
Regardless of the system, preparation requires casting two different layers of acrylamide
between glass plates. The lower layer (separating, or resolving, gel) is responsible for actually
separating polypeptides by size. The upper layer (stacking gel) includes the sample wells. It is
designed to sweep up proteins in a sample between two moving boundaries so that they are
compressed (stacked) into micrometer thin layers when they reach the separating gel.

Davidson College: SDS-PAGE

SDS-PAGE (PolyAcrylamide Gel

Electrophoresis)
The purpose of SDS-PAGE is to separate proteins according to their size, and no
other physical feature. In order to understand how this works, we have to
understand the two halves of the name: SDS and PAGE.

SDS
Since we are trying to separate many different protein molecules of different
shapes and sizes, we first want them denatured so that the proteins no longer have
any secondary, tertiary or quaternary structure (i.e. we want them to retain only
their primary amino acid structure). Consider two proteins that are each 500 amino
acids long but one is shaped like a closed umbrella whle the other one looks like an
open umbrella. If you tried to run down the street with both of these molecules
under your arms, which one would be more likely to slow you down, even though

they weigh exactly the same? This analogy illustrates mass and the 3D structure of
a molecule will determine how well it can move through an environment. We use
SDS to denature all proteins to the same linear shape.

Figure 1. This cartoon depicts what happens to a protein (pink line) when it is
incubated with the denaturing detergent SDS. The top portion of the figure shows a
protein with negative and positive charges due to the charged R-groups in the
protein. The large H's represent hydrophobic domains where nonpolar R-groups
have collected in an attempt to get away from the polar water that surrounds the
protein. The lower diagram shows that SDS can disrupt hydrophobic areas and coat
proteins with many negative charges which overwhelms any positive charges the
protein had due to positively charged R-groups. The resulting protein has been
denatured by SDS (reduced to its primary structure) and as a result has been
linearized.

SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic
molecules but also has a negative charge (sulfATE) attached to it. Therefore, if a
cell is incubated with SDS, the membranes will be dissolved, all the proteins will
be solubilised by the detergent, plus all the proteins will be covered with many
negative charges. So a protein that started out like the one shown in the top part of
figure 1 will be converted into the one shown in the bottom part of figure 1. The
end result has two important features: 1) all proteins retain only their primary
structure and 2) all proteins have a large negative charge which means they will all
migrate towards the positve pole when placed in an electric field. Now we are
ready to focus on the second half - PAGE.

PAGE
If the proteins are denatured and put into an electric field, they will all move
towards the positive pole at the same rate, with no separation by size. So we need
to put the proteins into an environment that will allow different sized proteins to
move at different rates. The environment of choice is polyacrylamide, which is a
polymer of acrylamide monomers. When this polymer is formed, it turns into a gel
and we will use electricity to pull the proteins through the gel so the entire process
is called polyacrylamide gel electrophoresis (PAGE). A polyacrylamide gel is not
solid but is made of a laberynth of tunnels through a meshwork of fibers (figure 2
and figure 3).

Figure 2. This cartoon shows a slab of polyacrylamide (dark gray) with tunnels
(different sized red rings with shading to depict depth) exposed on the edge. Notice
that there are many different sizes of tunnels scattered randomly throughout the
gel.

Figure 3. This is a top view of two selected tunnels (only two are shown for clarity
of the diagram). These tunnels extend all the way through the gel, but they
meander through the gel and do not go in straight lines. Notice the difference in
diameter of the two tunnels.

Now we are ready to apply the mixture of denatured proteins to the gel and apply
the current (figure 4). If all the proteins enter the gel at the same time and have the
same force pulling them towards the other end, which ones will be able to move
through the gel faster? Think of the gel as a tiny forest with many branches and
twigs throughout the forest but they form tunnels of different sizes. If we let
children and adults run through this forest at the same time, who will be able to get
through faster? The children of course. Why? Because of their small size, they
move through the forest faster since they have access to more of the paths in the
forest while adults are limited to only the larger paths. Likewise, small molecules
can maneuver through the polyacrylamide forest faster than big molecules.

Figure 4. Cartoon showing a mixture of denatured proteins (pink lines of different

lengths) beginning their journey through a polyacrylamide gel (gray slab with
tunnels). An electric filed is established with the positive pole (red plus) at the far
end and the negative pole (black minus) at the closer end. Since all the proteins
have strong negative charges, they will all move in the direction the arrow is
pointing (run to red).

You have to remember that when we work with proteins, we work with many
copies of each type of protein. As a result, the collection of proteins of any given
size tend to move through the gel at the same rate, even if they do not take exactly
the same tunnels to get through. Back to our analogy of the forest... If we were in a
hot air balloon above the forest and watched 100 children, 100 teenagers, and 100
large adults running through the forest, we would see a collection (or band) of
children moving quickly though each individual would choose his or her own route
through the forest. Behind the smallest individuals, we would see a band of
teenagers moving slower, and a third band made of adults plodding their way
through the forest using only the largest of paths. Likewise, proteins tend to move
through a gel in bunches, or bands, since there are so many copies of each protein
and they are all the same shape and size. When running an SDS-PAGE, we never
let the proteins electrophorese (run) so long that they actually reach the other side
of the gel. We turn off the current and then stain the proteins and see how far they
moved through the gel (until we stain them, they are colorless and thus invisible).
Figure 5 shows a cartoon gel and figure 6 shows a real one. Notice that the actual
bands are equal in size, but the proteins within each band are of different sizes.

Figure 5. Top view of an SDS PAGE after the current has been on for a while
(positive pole at the bottom) and then turned off. The gel (gray box) has five
numbered lanes where five different samples of proteins (many copies of each kind
of protein) were applied to the gel. (Lane 1, molecular weight standards of known
sizes; Lane 2, a mixture of three proteins of different sizes with a being the largest
and c being the smallest protein; Lane 3, protein a by itself; Lane 4, protein b by
itself; Lane 5 protein c by itself.) Notice that each group of the three proteins
migrated the same distance in the gel whether they were with other proteins (lane
2) or not (lanes 3-5). The molecular weight standards are used to measure the
relative sizes of the unknow proteins (a, b, and c).

Figure 6. This photo shows a variety of different proteins being separated on a gel.
This particular image is showing a serial dilution of the same protein sample to
indicate how little protein is needed (16 picograms = 16 . 10 -12 grams) in order to
be detected.
This image was taken from a home page operated by Hitachi Software.

There is a caveot to this method that you must always keep in mind. SDS-PAGE
separates proteins based on their primary structure or size but not amino acid
sequence. Therefore, if we had many copies of two different proteins that were
both 500 amino acids long, they would travel together through the gel in a mixed
band. As a result, we would not be able to use SDS-PAGE to separate these two
proteins of the same molecular weight from each other.

See Protein Gel with Stain

Methods Page

Copyright 2001 Department of Biology, Davidson College, Davidson, NC

28036
Send comments, questions, and suggestions to: macampbell@davidson.edu