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  • Quantitative Determinations of Proteins

    The quantitative estimation of proteins is one of the basic requirements
    in biochemistry. In reviewing the biochemical literature for
    methods of fast and sensitive determination of the amount of protein,
    the large variety of proteins becomes evident, since theamount
    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 protocolsmay 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 heterogeneous
    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 fromthe 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 estimated
    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.

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