Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Sample mechanical transport means in or for automated...
Reexamination Certificate
1998-02-10
2001-06-05
Alexander, Lyle A. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Sample mechanical transport means in or for automated...
C422S105000, C436S008000, C436S015000, C436S088000
Reexamination Certificate
active
06241946
ABSTRACT:
FIELD OF THE INVENTION
The present invention lies in the field of protein chemistry.
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
The present invention fills this need by providing for a solventless, protein-assay standard comprised of a receptacle, said receptacle containing a solventless dye and a dehydrated protein wherein upon addition of an appropriate solvent, the dye and protein react together to produce a detectable color change indicating the presence and/or amount of protein.
The present invention further provides for a method for preparing a solventless protein standard comprising:
preparing a dye solution containing a dye and a dye solvent;
placing an aliquot of the dye solution within a receptacle;
removing the dye solvent; preparing a protein solution containing a protein and protein solvent;
placing an aliquot of the protein solution within the receptacle containing the dye; and
removing the protein solvent such that the receptacle contains a solventless dye and a solventless protein.
A protectant or stabilizer such as anhydrous glycerol can be added to the solventless mixture. Generally, the protectant is placed between the dye and the protein.
Alternatively, the order can be reversed such that the aliquot of the protein solution can be placed into the receptacle first; the solvent removed and then the dye solution can be placed into the receptacle and the solvent removed.
REFERENCES:
patent: 4023933 (1977-05-01), Bradford et al.
patent: 4219337 (1980-08-01), Grossberg et al.
patent: 4305721 (1981-12-01), Bernstein
patent: 4337064 (1982-06-01), Gindler
patent: 4507233 (1985-03-01), Saito et al.
patent: 4859421 (1989-08-01), Apicella
patent: 4952516 (1990-08-01), Matkovitch
patent: 5073341 (1991-12-01), Hargreaves
patent: 5077222 (1991-12-01), Lau
patent: 5132085 (1992-07-01), Pelanek
Read & North cote, Anal. Biochem., 116:53-64(1981).
Compton & Jones, Anal. Biochem, 151:369-374 (1985).
Alexander Lyle A.
Lunn, Esq. Paul G.
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