western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students
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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 through 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,
1. PubMed: comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview.
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. 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.
The first step in the western blot protocol is membrane blocking (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).
•
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).
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.
• 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.