Biomedical Engineering Reference
In-Depth Information
1 Quantitative Methods
1.1 Quantitative Determinations of Proteins
The quantitative estimation of proteins is one of the basic require-
ments in biochemistry. In reviewing the biochemical literature for
methods of fast and sensitive determination of the amount of pro-
tein, the large variety of proteins becomes evident, since the amount
of protocols for quantitative protein seems to be innumerable.
Proteins, from many points of view, are much more complex
than, for example, nucleic acids. As a result, it has been difficult to
give laboratory protocols that can be applied to proteins in general;
however, in most cases the specialized protocols may be reduced to
a few basic methods. But if a protein becomes pure or some of its
unique properties are of special interest, another analytical method
has to be used. Nevertheless, accurate quantitation of the amount
of protein during the steps of protein preparation is the only valid
way to evaluate the overall value of a procedure.
The following protocols are based on distinct properties of
proteins; therefore, exact information is only possible if a hetero-
geneous protein mixture is compared with a universal standard
protein. The best way would be to take a defined sample of the
protein to be analyzed. So the difficulties start with the selection of
the standards, because it is well known how difficult it is to prepare
a protein that fulfills the criteria of analytical chemistry.
It is very often observed that during a purification process the
differences increase between the real amounts of a protein and the
values obtained by any method, e.g., total enzyme activity, because
the measured signal produced by a protein mixture differs from
that of a pure protein. Furthermore, the amount of a given protein
determined by a distinct protocol differs from the expected amount
by portioning, as shown in Table 1.1. To avoid additional mistakes
with the already uncertain process, the protein estimation method
should not be changed during a purification process.
With these difficulties kept in mind, any protein may be esti-
mated by one of the given protocols. Absolute statements, such as
“… the prepared, pure product has a specific activity of … units
per milligram of protein …” should be made with caution.
