Gel electrophoresis is a
technique which separates macromolecules - nucleic acids or proteins- on
the basis of size, electric charge and other physical properties.
SDS-Polyacrylamide gel electrophoresis (PAGE) further resolves complex
mixtures of proteins by using the ability of a detergent SDS (sodium
dodecyl sulfate) to denature and reduce polypeptides to flexible rods
with uniform negative charge per unit length.
Since molecular weight is now a linear function of peptide length, the
proteins separate by molecular weight through the pores of the gel. The
smaller the molecule, the faster it separates. The purpose is to separate or isolate proteins from a crude
sample, estimate the purity and the molecular weight of a protein in
kilodaltons (kDa).
The technique
Electrophoresis is the migration of charged
molecules in solution inside a support matrix (a colloid in a solid
form) subjected to an electrical
field (between a negative and a positive electrode). Here,
polyacrylamide gel will be used as a support matrix: it is used to
separate most proteins and small oligonucleotides requiring a small gel
pore size. Polyacrylamide gel is polymerized from a solution of
acrylamide monomers into cross-linked chains, forming a semi solid
matrix.
The rate at which the proteins move in the gel depends on the electric
current, the pH of the medium, the pore size of the gel, the friction
which in turn depends on the shape and size of the protein
(smaller molecules will move faster through the pores of the gel).
Considerations over the separation of
proteins:
The net charge of a protein depends on the pH of its environment. In a
solution with a pH above its isoelectric point, a protein has a net
negative charge and will migrate towards the positive electrode. Below
the isoelectric point, the protein is positively charged and therefore
migrates towards the negative electrode. It is important to note that
the net charge carried by a protein is also independent of its size: the
charge carried per unit mass of molecule is different from protein to
protein.
At a given pH and under non-denaturing conditions, the electrophoretic
migration of proteins is determined by size and charge of the molecules.
If the matrix is buffered to weakly
basic, most proteins loaded on the gel will be anionic and migrate to
the positive electrode when an electric current is applied.
If one chooses to limit the migration
of proteins to only one factor, namely protein size or mass, then
further treatment of the proteins by an anionic detergent (sodium
dodecyl sulfate or SDS) must be considered.
Migration of proteins under
denaturing conditions:
SDS is an ionic detergent with a long hydrophobic tail which binds to
the hydrophobic side chains of aminoacids at a constant ratio of 1.4 g
of SDS per gram of polypeptide. This ratio ensures that all SDS treated
proteins have a similar mass to charge ratio; so, net protein charge is
no longer a factor affecting its migration. SDS also denatures proteins,
dissociates multimeric proteins into subunits and causes
extended conformations ("flexible rods") in the polypeptides. As a result, shape of
the protein is no longer a factor in its migration, but its molecular
weight is.
The
picture above shows
gels that have been mounted in one electrophoresis apparatus and
which will receive
samples for protein migration and separation. Special long tips
are used to facilitate loading.
The comb is gently removed to allow the running buffer
to enter the wells. The samples
are loaded slowly, into separate wells, with the pipette tip close to the bottom of
the well without touching the gel.
Result
The proteins, stained with a
fluorescent dye, are loaded into the wells of the gel and
electrophoresed. Bands appear on the gel, showing the migration
distances of different sized proteins. Migration distances are
characteristic to specific proteins and are used to assess the purity of
protein. If the protein under study is unidentified, it can be compared
to the migration distances of a known protein (standard) for an estimation of its
molecular weight. The gel concentration must be chosen so that the
standards give out a linear curve in the region of the unknown.
For an interesting
electrophoresis simulation from the Rochester Institute of Technology,
click here
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