Colorimetric protein quantitation assays

TABLE 3 Common Colorimetric Protein Quantitation Assays... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

Finally, there are custom two-step quantitation methods such as chromatography or ELISA that require a capture step for isolating the protein and then a quantitation step based on a standard curve of the purified target protein. The preliminary capture step may also concentrate the protein for increased sensitivity. These techniques are typically not available in a commercial kit form and may require extensive method development. They are more labor intensive and complex than the colorimetric or absorbance-based assays. In addition, recovery of the protein from and reproducibility of the capture step complicate validation. Despite these disadvantages, the custom two-step quantitation methods are essential in situations requiring protein specificity. 

The first assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein. Amb a 1 is approximately 1% of the total protein extracted from ragweed pollen hence the Bradford assay does not reflect Amb a 1 concentration. However, at this step of the production process, the protein concentration is used to calculate final yields and not to make time-dependent or expensive decisions. Hence the nonspecific Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

The four colorimetric methods for the detection and quantitation presented in this unit have withstood the test of time. They are all well-characterized robust assays that consistently work well. The methods were introduced over the past 15 to 50 years. They collectively represent the state of the art for colorimetric detection and quantitation of total proteins in the microgram to milligram range. 

In 1976, Marion Bradford introduced the first Coomassie dye-based reagent for the rapid colorimetric detection and quantitation of total protein. The Coomassie dye (Bradford) protein assay reagents have the advantage of being compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples. 

In this unit, Basic Protocol 1 presents a procedure using casein as substrate. The Alternate Protocol describes the modification of this procedure for use with a denatured hemoglobin substrate. Basic Protocol 2 presents a procedure using a chromaphore-conjugated casein derivative, azocasein. For quantitation, the authors have chosen to use either the BCA-based colorimetric assay unitbli) for soluble protein/peptides (in Basic Protocol 1) or the intrinsic absorbance of the chromaphore-conjugated peptide products (in Basic Protocol 2). 

Another method consists of a spectroscopic measurement of Comassie brilliant blue stained SC protein directly on the tape.17 In contrast with the colorimetric method described earlier, this method does not require any SC extraction procedure prior to protein assay. However, it has been shown to be variable and not appropriate for quantitative determination since the absorbance of colored SC proteins is negligible as compared to light scattering of the SC material adhering to tape strips. 

As mentioned above, the large majority of recoverable amino acids are in combined form. This result indicates that peptides, and possibly proteins, are important DON constituents. Operational measurements of the amount of total protein in DON can be made by a wide variety of fluorometric and colorimetric assays. A partial hst of those that have been applied to marine samples include coomassie blue (e.g., Mayer et at, 1986 Nunn et ai, 2003 SetcheU, 1981), Lowry s method (Clayton et al., 1988), the fluorescamine assay (Garfield et al., 1979), the bidnchoninic acid assay (Nguyen and Harvey, 1994), and the CBQCA (3 -carboxybenzoyl-quinoline-2-carboxaldehyde) assay (Nunn et al., 2003). A number of the more common methods of protein analysis used in biochemical research, including a discussion of issues affecting their quantitative apphcation, have recently been reviewed by Sapan et al. (1999). 

Total protein assays have the advantage of being relatively straightforward compared to molecular-level analyses. Methods with fluorescence-based detection are also highly sensitive, and thus amenable direcdy to DON. Quantitative interpretation for environmental mixtures such as seawater, however, may be problematic for some samples. Most methods react with specific moieties (e.g., coomassie blue binds to lysine and arginine) and thus results obtained can depend on protein composition, size distribution, and even conformation (Sapan et ai, 1999), making the careful choice of calibration standards important. In addition, common components of natural samples, such as humic materials (e.g., Mayer et ai, 1986), carbohydrates (Sapan et ai, 1999), or NH3 may interfere with quantification. Overall, colorimetric methods can be very useful as quick, Hkely semi-quantitative estimates of total protein or peptide. However, potential biases inherent in the mechanism of a specific method should be considered before one is chosen, and appHcation of newer molecular assays (e.g., CBQCA) should be carefully examined in terms of natural sample matrix (Nunn et ai, 2003). 

Heisler, I., Keller, J., Tauber, R., Sutherland, M. and Fuchs, H. (2002) A colorimetric assay forthe quantitation of free adenine applied to determine the enzymatic activity of ribosome-inactivating proteins. Anal Biochem, 302, 114-122. 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

Quantitative analysis of proteins can be achieved by UV spectroscopy. The peptide bond has an absorption maximum around k = 205 nm, the aromatic rings on the amino acids Tryptophan and Tyrosine absorb strongly around k = 280 nm. Also commonly used are colorimetric assays, which contain reagents that specifically form coloured complexes with proteins. These quantitative methods usually measure the total protein concentration. Either the protein of interest has to be isolated prior to analysis, or a very specific method has to be found to quantify only the targeted protein. Very sensitive and specific analysis of antibodies and antigens can be achieved with bioassays (section 5.1) or biosensors (section 5.2). 

Effect of Ascorbate on Cell Metabolism. We addressed the question of whether ascorbate-induced suppression of RT and p24 production in H9/HTLV-IIIb cells was a virus-specific effect or an indirect effect due to inhibition of cellular metabolism or protein synthesis. The metabolic activity of uninfected H9 cells in the presence and absence of ascorbate was determined by using a quantitative colorimetric assay that utilizes the tetrazolium salt MTT (18). MTT is used to measure the activity of various dehydrogenases in viable cells (18, 19). H9 cells grown in the presence of various concentrations of ascorbate (0-150 /tg/ml) showed an increase in cellular metabolic activity on day 1 (Fig. 4). This correlated with stimulation of cell proliferation by ascorbate. On days 2 and 4, no significant change in metabolic activity was noted between control cultures and those exposed to ascorbate at 75,100, and 150 /xg/ml. 

Recent advances have enormously widened the range of applicability of amino acid determinations as a control on fractionation. Not only have colorimetric procedures been revised and increased in accuracy, but the development of microbiological and chromotographic methods of assay now permits the quantitative determination of almost any amino acid residue, using very small amounts of protein of the order of 10 to 100 mg. The results are rapid and, by some at least of tiiese methods, appear to be trustworthy. Since the general principles of all these methods have been reviewed in the previous volume of this series, by Martin and Synge (142) and by Snell (197), no further discussion of them need be given here. 

The concentration of a protein can be determined by various colorimetric methods including the old standard Folin—phenol reagent assay of Lowry (79) or the new standard Coomassie blue R-250 method of Bradford (80) or bicin-choninic acid (BCA) assay. Silver stain (108) and gold stain are also widely used to identify and quantitate proteins with a 10-50 times greater sensitivity than Coomassie blue staining. Once a reliable estimate of the protein concentration is obtained, the UV absorptivity of the known protein can be used to determine unknown concentrations of the same protein. From the Beer—Lambert law, A(x) = o% lJI) = where is the absorbance (or optical density) at the... 

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