Array of Assays

Protein Assay Manufacturers and Products From the decades-old biuret reaction to the use of complicated fluorescent molecules, the search for an easy, reliable, and stable method for the quantitation of total protein in solution has gone on for more than 80 years. Although several famous, innovative, and useful techniques were developed over that time, the search continues. Bob Vigna, marketing manager for industry leader Pierce Chemical Co. of Rockford, Ill., confirms this observation. "The use of protein assays is still a growing area for us," he says. Pierce has recently introduced its new Coomassie Dry Protein Assay Plate. The first protein assay available prepared in a ready-to-use 96-well plate format, the Coomassie Dry Assay uses a unique Bradford formulation stabilized as a glass within each well. Samples are simply added, mixed, and read on a microplate reader. Typically, the conventional protein assay consists of the addition of a chemical agent to the protein sample. A visual colored product is created, which is usually measured with a spectrophotometer. When compared to a standard curve prepared with known concentrations of a purified protein, the amount of protein in the unknown sample can be extrapolated based on its absorbance. Besides their standard forms, many assays have a micro version designed for smaller or dilute samples, in addition to microplate formats. The types of chemical agents used to detect protein form the basis of the individual assays developed and commercialized throughout the years. The three standard and well-known methods, the Lowry, bicinchoninic acid (BCA), and Bradford assays are seeing competition from newer assays based on novel detection techniques that include fluorescent molecules, colloidal gold, and some proprietary compounds. Geno Technology's Non-Interfering Protein Assay The Lowry Assay As the most frequently cited protein assay in the literature, the venerable Lowry assay is the standard to which most other protein assays are compared.1 After its development in 1951, the Lowry assay became the most widely used procedure for the quantitative determination of protein and maintains a loyal following. Although subject to intense scrutiny and numerous modifications throughout the years,2 the assay nevertheless remains a benchmark technique in the annals of life science research. The Lowry technique begins with the "biuret" reaction in which a protein in an alkaline system containing Cu2+ forms a colored complex when the copper atoms are reduced to Cu1+ through a reaction with the peptide bonds. In fact, the biuret reaction itself had previously been used for many years to quantify proteins in solution, but it was hampered by low sensitivity. Lowry and coworkers Rosebrough, Farr, and Randall improved the biuret reaction's sensitivity with the addition of the Folin phenol reagent of Folin and Ciocalteau. The active constituents of the Folin-Ciocalteau reagent, phosphomolybdic/phosphotungstic mixed acids, are reduced by the copper-treated protein, generating an intense blue color that can be measured at 750 nm. Despite its acceptance, the traditional Lowry assay has specific disadvantages, such as reagent instability and the requirements for exact timing of reagent addition and mixing to obtain accurate results. Consequently, modifications of the Lowry assay have found their way into the commercially available Lowry-type assay products. Pierce Chemical's Modified Lowry Protein Assay, for example, is based on a modification that replaces two of the traditional Lowry reagents with one stable solution.3 The result is a stable, preformulated product with nearly 100 percent color response correlation with the original Lowry assay. This assay has a working range of 1 to 1500 µg/ml. The Bio-Rad DC Protein Assay from Bio-Rad Laboratories Inc. of Hercules, Calif., is a modified Lowry assay developed for protein samples solubilized in detergent. Capable of functioning in the presence of 1 percent detergent and many other common reagents, the assay requires only a 15-minute incubation period before samples can be read. Absorbances are stable for at least two hours. In September, Bio-Rad introduced the RC DC Protein Assay, a colorimetric assay for determining protein concentration in the presence of both reducing agents and detergents. For example, the RC DC Protein Assay can readily quantitate protein in a Laemmli Buffer/ß-Mercaptoethanol solution. Based on a modification of the Lowry protocol, this new product has all the unique capabilities of the original DC Protein Assay, in addition to covering a broader range of reagents. Sigma-Aldrich of St. Louis offers two modified Lowry assays. The Modified Lowry Kit for Protein Determination is also based on the above-mentioned modification.3 The kit facilitates lipoprotein dissolution and uses deoxycholate and trichloroacetic acid to precipitate proteins away from interfering substances. Additionally, the Micro Modified Lowry Kit for Protein Determination has been developed for assaying proteins in biological fluids. The BCA Assay Reasoning that most difficulties associated with the original Lowry assay are related to the use of the Folin-Ciocalteau reagent, researchers at Pierce Chemical developed an alternative assay using BCA as a replacement. BCA, a sensitive, stable, and highly specific reagent for Cu1+, forms a purple-colored product when it reacts with the Cu1+ produced by the biuret reaction.4 In comparison to the Lowry assay, the BCA assay is easier to use, has similar protein-to-protein variation, has improved reagent stability, and possesses a broader tolerance to interfering compounds. In particular, the BCA assay tolerates detergents and denaturing agents such as 4 M guanidine-HCl and 3 M urea. The resulting BCA Protein Assay is ideal for measuring detergent-solubilized membrane proteins and has a working range of 20 to 2,000 µg/ml. For dilute protein samples, Pierce offers the Micro BCA™ Protein Assay, containing a higher concentration of BCA than the original version and using a modified protocol. The assay is compatible with detergents and exhibits less protein-to-protein variability than the BCA, Bradford, or Lowry assays. Finally, Sigma-Aldrich carries a BCA protein assay called the Bicinchoninic Acid Kit for Protein Determination. Sigma-Aldrich's Protein Assay Kits and reagents The Bradford Protein-Dye Binding Assay A workhorse protein assay used by thousands of labs, the protein-dye binding assay created by Marion Bradford utilizes the color change that occurs when Coomassie Blue G-250 binds to proteins in an acidic solution.5 The absorbance maximum of Coomassie Blue G-250 shifts from 465 to 595 nm on binding, forming the characteristic blue-colored compound readily measured by a spectrophotometer. The Bradford assay is fast and convenient, and its characteristics and limitations are well-known and understood. In particular, the dye-binding response of the assay varies from protein to protein and tends to generate nonlinear standard curves. The assay is sensitive to detergents, and it is compatible with reducing agents such as DTT and ß- mercaptoethanol. The standard procedure calls for adding the acidified Coomassie Blue G-250 solution (Bradford reagent) to the protein sample, mixing, and then measuring the absorbance after a five-minute incubation period. The oldest commercially available version of the Bradford assay, and perhaps the most widely known, is the Bio-Rad Protein Assay from Bio-Rad Laboratories. With a working range of 0.2 to 1.4 mg/ml, the assay requires only five minutes for preparation and is readily adaptable to a microplate format. In addition to the Coomassie Dry Protein Assay Plates mentioned above, Pierce Chemical offers two versions of the Bradford assay. The first, the Coomassie Protein Assay Reagent, consists of a ready-to-use formulation of dye and demonstrates all of the typical characteristics of the published Bradford method. Pierce's second Bradford assay, the Coomassie Plus Protein Assay Reagent, contains an advanced ready-to-use formulation of the Bradford Coomassie Blue G-250 reagent. Here, the modified Bradford reagent gives a more linear response than the typical Bradford formulation and demonstrates about half of the protein-to-protein variation seen with other Bradford-type assays. Amresco, Geno Technology, and Sigma-Aldrich also offer Bradford protein assays. Pierce's BCA™ Protein Assay Running Interference Interfering substances and protein-to-protein variability are the major drawbacks to the traditional protein assays. Many of the biochemical reagents commonly used with proteins in solution, such as detergents, reducing agents, and buffer salts, can artificially enhance or attenuate the color response of an assay. Often, these substances can overwhelm an assay, making it unusable; the list of substances that interfere with the Lowry assay is extensive, although to be fair, no other assay has been as thoroughly examined.2 Variations among proteins stemming from their amino acid sequences, side chains, isoelectric points, or structures often result in altered responses depending on the assay and its particular mechanism. To illustrate, a comparison of the estimated protein concentrations for 23 different purified proteins as determined by the Coomassie Blue (Bradford) and Lowry assays demonstrated that the Coomassie Blue assay had twice the standard deviation as the Lowry.2 This variation can cause difficulties when the protein being assayed responds differently than the protein used to prepare the standard curve, or when comparing assays of separate proteins. Though numerous methods have been suggested to address these drawbacks of the traditional assays, the desire for protein assays that are free from interference by common reagents and that exhibit little protein-to-protein variation remains high. A few assays, developed and released in the last few years, now address these issues. One of the newer protein assays, developed by Cytoskeleton Inc. of Denver, Co., is the Advanced Protein Assay Reagent. This product grew out of a strong need for an assay with low variance between proteins, according to Ashley Davis, head of new-product development for Cytoskeleton. "We manufacture over 150 purified proteins. We needed one assay that worked effectively with all of them, so we developed our own," Davis explains. Indeed, for 95 percent of the individual proteins assayed with the Advanced Protein Assay Reagent, the estimated concentrations fall within 20 percent of the true value. The assay procedure involves one step: A small amount of the protein sample is added to the reagent and mixed by vortexing for five seconds. The color shift from emerald green to marine blue is measured at 570 to 615 nm. In addition, the color change is striking enough to allow semiquantitative estimations to be made visually by comparison to a color-coded chart. Another system, the Non-Interfering Protein Assay™ from Geno Technology of Maplewood, Mo., has been extensively tested to work with reducing agents, chelating agents, detergents, amines, and many other compounds. "I wish I had something like this years ago," says Aftab Alam, president of Geno Technology. In fact, extraction buffers containing mixtures of reagents such as guanidine thiocyanate, EDTA, and ß-mercaptoethanol will not affect it. The strategy employed by the Non-Interfering Protein Assay first involves the removal of the protein from the interfering compounds using Geno Technology's proprietary Universal Protein Precipitating Agent, which rapidly precipitates and immobilizes proteins within the sample tube. Proteins are then detected based on the specific binding of copper ions to the peptide backbone. Unbound copper reacts with a reagent to produce a light orange color that is measured spectrophotometrically. The color is inversely related to the amount of protein, and, because the technique is independent of amino acid side chains, protein-to-protein variation is dramatically reduced. Using an optimized version of the biuret reaction,6 the Protein Assay ESL (Exact, Sensitive, Low Interference) from Roche Molecular Biochemicals of Indianapolis offers minimal protein-to-protein variability and broad tolerance to interfering substances. This method starts with a biuret reaction in which Cu2+ initially complexes with protein in an alkaline solution. Excess Cu2+ not chelated by the protein is reduced to Cu1+ with ascorbic acid. The Cu1+ then forms a colored complex with bathocuprine. As a result, the signal measured on the spectrophotometer is inversely related to the amount of protein. Protein-to-protein variations are low because the copper reacts with the proteins via the peptide bonds rather than specific amino acid side chains that vary from protein to protein. The Protein Assay ESL is linear between 20 and 800 µg per ml and is also suitable for peptides and immobilized proteins. Fluorescence Representing a departure from the typical colorimetric protein assay, Molecular Probes Inc. of Eugene, Ore., has developed two unique fluorometric methods for quantifying proteins in solution. Touted as out-performing all existing methods in terms of sensitivity and effective range, the two kits detect as little as 10 ng of protein with linear dynamic ranges that extend across three orders of magnitude. The NanoOrange Protein Quantitation Kit uses the fluorescent reagent NanoOrange to detect proteins at a sensitivity level 50 to 100 times better than the Lowry, BCA, or Bradford assays.7 Though the assay is sensitive to some of the common reagents found in protein preparations, it is insensitive to reducing agents and exhibits little protein-to-protein variability. Molecular Probes' CBQCA Protein Quantitation Kit employs ATTO-TAG™ CBQCA reagent, a compound developed primarily to detect attomole (10-18) levels of amino acids, peptides, and proteolytic fragments by amine derivatization in capillary electrophoresis.8 The ATTO-TAG CBQCA reagent is essentially nonfluorescent in an aqueous solution. In the presence of cyanide, however, it reacts with the primary amines found on proteins to form highly fluorescent derivatives. The CBQCA assay works well in the presence of lipids and detergents. Gold and Spots Motivated by the discovery that colloidal gold detects nanogram levels of proteins on western blots, C.M. Stoscheck developed a rapid and sensitive protein assay that also capitalizes on the affinity of colloidal gold for protein.9 Capable of detecting as little as 5 ng of protein in solution, Stoscheck's method, with a subsequent modification,10 forms the basis of the Quantigold Protein Assay from Diversified Biotech of Boston. Extremely sensitive, Quantigold requires only nanogram amounts of protein for quantification. In operation, the assay is linear up to 200 ng and reliable to 500. The assay is simple: 10 µl samples are added to 800 µl aliquots of the Quantigold reagent, incubated at 37°C for 40-60 minutes, and then measured spectrophotometrically. The assay is also very stable, being accurately read up to 24 hours after its completion. Offering an unusual approach to protein quantitation, the dotMETRIC™ Protein Assay from Geno Technology quantitates proteins based on the diameter of protein sample spots formed on special test strips. One µl protein samples are spotted onto the test strips, which are then developed by sequential incubation in two reagents. High contrast spots appear in less than 10 minutes. The diameter of the spot, as measured by the dotMETRIC gauge, is proportional to the protein concentration. Work with various proteins has demonstrated that the diameter of the spot is not dependent on the nature and origin of the protein. In addition, most common laboratory reagents do not interfere with the assay. Because the accuracy of the test can depend on the pipetting skills of the user, however, Geno Technology now offers a new version of the kit containing a dotMETRIC Spot Application Device and fixed 1 µl glass capillary application tips. This device produces uniform and reproducible spots, eliminating person-to-person variability. With so many types and variations of protein assays available, the choice of an appropriate assay becomes a difficult task. Things to consider include the type and amount of protein available, the chemical composition of the buffer, potential interference with the assay, and access to appropriate instrumentation such as a fluorometer or microplate reader. Most of the manufacturers provide extensive lists of the reagents that are compatible or incompatible with their assays. Contact technical service departments with specific questions. S Michael D. Brush (michaeldbrush@cs.com) is a freelance writer in Orange, Calif. References 1. O.H. Lowry et al., "Protein measurement with the Folin phenol reagent," Journal of Biological Chemistry, 193:265-75, 1951. 2. G.L. Peterson, "Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall," Analytical Biochemistry, 100:201-20, 1979. 3. G.L. Peterson, "A simplification of the protein assay of Lowry, et al. which is more generally applicable," Analytical Biochemistry, 83:346-56, 1977. 4. P.K. Smith et al., "Measurement of protein using bicinchoninic acid," Analytical Biochemistry, 150:76-85, 1985. 5. M.M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding," Analytical Biochemistry, 72: 248-54, 1976. 6. M. Matsushita et al., "Determination of proteins by a reverse biuret method combined with the copper-bathocuprine chelate reaction," Clinica Chemica Acta, 216:103-11, 1993. 7. L.J. Jones et al., "New fluorescent assay for detection and quantitation of nanogram levels of proteins in solution," FASEB Journal, 10:A1512 (Abstract #2954), 1996. 8. W.W. You et al., "3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, a reagent with broad dynamic range for the assay of proteins and lipoproteins in solution," Analytical Biochemistry, 244:277-82, 1997. 9. C. M. Stoscheck, "Protein assay sensitive at nanogram levels," Analytical Biochemistry, 160:301-5, 1987. 10. T. Ciesiolka, H. Gabius, "An 8- to 10-fold enhancement in sensitivity for quantitation of proteins by modified application of colloidal gold," Analytical Biochemistry, 168:280-3, 1988.

Array of Assays

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