DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 13

Western blot analysis (Immunoblotting)
Western blotting is a technique analogous to Southern and Northern
blotting (see Chapter 2), but for proteins. The method was first reported
in 1979 (7), following the description of the successful transfer proce-
dure for DNA developed by E.M. Southern. Since Southern blotting was
the first blotting method developed, the technique is presented in more
detail in that section. After fractionating proteins on polyacrylamide gels,
the proteins can be transferred quantitatively to membranes by capillary
action or by electroblotting. Capillary transfer is lengthy and highly ineffi-
cient (10-20% of the proteins transfer), whereas electroblotting is rapid
(2 to 3 hr) and efficient. In electroblotting, a membrane is placed next to
a gel and submerged in a buffer. An electric current is passed at right
angels to the gel, and the proteins are transferred onto the membrane.
The proteins that have been transferred to the membrane are then de-
tected by a probe, an agent that can selectively and specifically identify a
particular protein or related proteins. For western blot analysis, antibod-
ies are the most common probes. The antibodies are directly labeled with
a radioactive molecule, such as so that its binding is detectable by
autoradiography. Alternatively, it can be labeled with an enzyme, such
as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The
blot can then be treated with the appropriate substrate solution, which
is converted into an insoluble material which identifies the location of
the antibody bound to the protein of interest on the membrane. How-
ever, most laboratories now use enhanced chemiluminescence (ECL)
to detect the enzyme linked antibody (see Chapter 4).
There are a number of variations of the basic western blotting proce-
dure described above. One of the most common is to take advantage
of secondary antibodies. Secondary antibodies are “antibodies against
antibodies” that have been labeled with or an enzyme. The use of
secondary reagents negates the need to label primary antibodies. In
this way, only a small number of secondary antibodies need to be la-
beled, rather than all of the primary antibodies of interest. Alternatively,
biotinylated primary antibodies can be used. Biotin is a small molecular
weight vitamin that is strongly bound by avidin, a protein from egg, which
can be used as a secondary reagent rather than another antibody. This
system is discussed further in Chapter 4.
Chromatography
Chromatography is a generic term that applies to a variety of meth-
ods that separate macromolecules. Chromatography is a method that
takes advantage of columns (vertical tubes) that contain a matrix that is