Biomedical Engineering Reference
In-Depth Information
Figure 1. Two-dimensional polyacrylamide gel electrophoresis
second dimension under reducing conditions. Figure 1 shows cell ex-
tracts from metabolically-labeled plasma cells (which synthesize and
secrete antibodies; see Chapter 4) that have been fractionated on na-
tive gels in the first dimension, and then resolved in the second dimen-
sion under denaturing and reducing SDS-PAGE gels. The antibody pro-
duced by this cell is IgM which has many intracellular oligomeric forms as
shown in the native gel in the first dimension, and these can approach
sizes estimated to be 1 million Daltons. Using a reducing SDS-PAGE
second dimension, each of the oligomeric species can be shown to be
comprised of a typical IgM heavy chain, which is about 85,000 Daltons
(or 85 kiloDaltons, or kDa) and a light chain, estimated to be 23 kDa.
Molecular weight estimates are made by running “markers” of known
molecular weight in each gel. Chapter 4 provides more information on
the structure of antibodies and their function.
Detection of proteins in gels
It's great to be able to fractionate proteins in gels, but how do you see
them once the fractionation is completed? There are a number of ways
to detect proteins in gels. One of a number of stains can be used to
reveal their presence, autoradiography (exposure to photographic film)
can be used if the proteins have been metabolically labeled or had a
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