Description
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 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.