The determination of the total protein present within a solution is important in many analytical procedures, such as in the determination of the purity of a protein and in determining the protein chemistry of a blood or urine sample.
Four spectroscopic methods are routinely used to determine the concentration of protein in a solution. These include measurement of the protein's intrinsic ultraviolet (U.V.) absorbance, and three methods which generate a protein-dependent color change, namely, the Lowry assay, the Smith copper/bicinchoninic assay and the Bradford dye assay. Although one or more of these methods is used routinely in almost every biochemical laboratory, none of the procedures are particularly convenient for the reasons described below.
The first, U.V. absorbance, has limited application since for accuracy it requires a pure protein with known extinction coefficient in a solution free of interfering substances. The Lowry and copper/bicinchoninic assays require the preparation of several reagent solutions, which must be carefully measured and mixed during the assay. This is followed by lengthy, precisely timed incubations at closely controlled, elevated temperatures, and then immediate absorbance measurements of the unstable solutions. Both assays may be affected by other substances frequently present in biochemical solutions, including detergents, lipids, buffers and reducing agents. To control for these factors, every assay must also include a series of standards, each with a different, known concentration of added protein, but otherwise having the same composition as the sample solutions. The Bradford dye assay is faster, involves fewer mixing steps, does not require heating, and gives a more stable colorimetric response than the two previous assays. Like them, however, its response is quite prone to influence from nonprotein sources, and protein standard solutions are necessary.
The preparation of the protein standards is tedious and error-prone. Protein preparations, usually of bovine serum albumin, are available as preweighed powder or in sterile solutions of measured concentration, but their use in preparing standard solutions still requires several precise measurements and dilutions. Frequently this involves compromises between convenience, precision, and the requirement that the standards contain the same concentrations of non-protein components as the sample solutions. For many applications, during protein purification, for example, the protein concentrations of several different solutions need to be assayed. Unless a set of protein standards is prepared for each of the different solutions, the assay results may be wrong. Further, protein standard solutions are inherently unstable, and decompose at varying rates depending on factors which include their composition, pH, sterility and conditions of storage. This reduces the reliability of the standard solutions, and hence of the assay itself, and requires the frequent preparation of new standard solutions.
In conclusion, protein assays are inconvenient and sometimes unreliable due to the problems associated with the preparation of the necessary protein standards. A method which allowed protein assays to be performed without these problems would be of considerable value. Thus, there is a need for a protein assay standard in which the preparation of standard solutions is eliminated.
USPTO Backgrounds
USPTO Backgrounds