Gel
electrophoresis
SDS-PAGE (PolyAcrylamide Gel Electrophoresis)
is used to separate molecules based on size, shape, or isoelectric
point. SDS-PAGE, coupled with western blotting (immunoblotting)
is typically used to determine the presence and /or relative
abundance of a given protein.
The gel is a cross linked polymer matrix used to support and
separate the molecules. The gel density can be controlled
by varying the monomer concentration. Gels can be of constant
density or they can be variable (gradient gels). After cross
linking has taken place, the samples are loaded in small wells
in the gel and the assembly undergoes electrophoresis.
Electrophoresis involves applying an electric current to the
gel and allowing the proteins to migrate though the matrix.
In order for the proteins to migrate through the gel (figure
1), they are first denatured and negatively charged by exposure
to a detergent such as sodium dodecyl sulfate (SDS). The amount
of bound SDS is relative to the size of the protein, and the
proteins have a similar charge to mass ratio. Bands in different
lanes separate based on the individual components sizes. A
molecular weight marker that produces bands of known size
is used to help identify proteins of interest.
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Figure 1 - SDS-PAGE
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After
the protein components have been sufficiently separated by
electrophoresis, they can be transferred to a PVDF or nitrocellulose
membrane. The transfer process uses the same principle as
SDS-PAGE – this time the electric current is applied
at 90 degrees to the gel and the proteins migrate out of the
gel onto the membrane. Once the proteins are separated and
bound to the membrane support, western
blotting can begin.
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Figure 2 - Blocking the membrane
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Western
blotting is used to detect a target protein in a sample
(containing a complex mixture of proteins) by using a polyclonal
or monoclonal antibody specific to that protein.
Western
Blot Procedure
• Blocking
The membrane is blocked (figure 2), in order to reduce non-specific
protein interactions between the membrane and the antibody.
This is achieved by placing the membrane in a solution of
bovine serum albumin (BSA) or non-fat dry milk (NFDM).
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•
Primary Antibody
The first antibody to be applied (specific for protein of
interest) is incubated with the membrane. The antibody is
diluted in a buffer solution (PBS) containing a carrier protein
(BSA or NFDM) along with some detergent. The primary antibody
is specific for the protein of interest, and, at appropriate
concentrations, should not bind any of the other proteins
on the membrane (figure 3 – red antibody).
• Secondary Antibody
After rinsing the membrane to remove unbound primary antibody
a secondary antibody (figure 3 – green antibody) is
incubated with the membrane. It binds to the primary antibody.
This secondary antibody is typically linked to an enzyme that
allows for visual identification by producing fluorescence.
An alternative is to use a radioactive label.
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Figure 3 - Antibody - antigen interaction
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Developing
The unbound secondary antibodies are washed away, and the enzyme
substrate is incubated with the membrane so that the positions of
membrane-bound secondary antibodies will emit light. Bands corresponding
to the detected protein of interest will appear as dark regions
on the developed film. Band densities in different lanes can be
compared providing information on relative abundance of the protein
of interest.
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