Biomedical Engineering Reference
In-Depth Information
Well 2:
Purified
protein
Well 3:
Molecular mass
standards
Well 1:
Crude protein
mix
Polyacrylamide
slab gell
Mobility
through
gel (mm)
Log molecular mass
+
Anode
(a)
(b)
Figure 7.1
Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing
agents, which disrupt disulfi de linkages). The electric fi eld is applied across the gel after the protein samples
to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent
upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining
with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass,
a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation
of the molecular mass of the purifi ed protein
One concern relating to SDS-PAGE-based purity analysis is that contaminants of the same mo-
lecular mass as the product will go undetected as they will co-migrate with it. Two-dimensional
electrophoretic analysis would overcome this eventuality in most instances.
Two-dimensional electrophoresis is normally run so that proteins are separated from each other
on the basis of a different molecular property in each dimension. The most commonly utilized
method entails separation of proteins by isoelectric focusing (see below) in the fi rst dimension,
with separation in the second dimension being undertaken in the presence of SDS, thus promoting
band separation on the basis of protein size. Modifi ed electrophoresis equipment that renders two-
dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-
cal fi nished products to such systems allows rigorous analysis of purity.
Isoelectric focusing entails setting up a pH gradient along the length of an electrophoretic
gel. Applied proteins will migrate under the infl uence of an electric fi eld until they reach a
point in the gel at which the pH equals the protein's isoelectric point pI (the pH at which the
protein exhibits no overall net charge; only species with a net charge will move under the in-
fl uence of an electric fi eld). Isoelectric focusing thus separates proteins on the basis of charge
characteristics.
This technique is also utilized in the biopharmaceutical industry to determine product homo-
geneity. Homogeneity is best indicated by the appearance in the gel of a single protein band,
exhibiting the predicted pI value. Interpretation of the meaning of multiple bands, however, is
less straightforward, particularly if the protein is glycosylated (the bands can also be stained for
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