Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml⁻¹) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml⁻¹) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

Quantitation of yeast total proteins in sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer for uniform loading

Proteins in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS–PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays.     

Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents

A procedure was developed for the quantitation of solubilized proteins using the Bradford assay in the presence of glucopyranoside detergents. These detergents solubilized membrane-bound proteins with minimal background absorbance at 595 nm. Absorbance at 650 nm was also low, indicating that these detergents do not significantly stabilize the neutral species of Coomassie brilliant blue G-250 that produces interference in the presence of other detergents. Hexyl-beta-D-glucopyranoside produced less absorbance than did larger glucopyranosides, and the increase in its absorbance at 595 nm in the presence of dye reagent was related linearly to its concentration from 0 to 2%. Absorbance produced by membrane-bound protein was increased by the presence of up to 0.2% hexyl-beta-D-glucopyranoside (final concentration in dye reagent) and then remained stable up to 1%, indicating that these concentrations of this detergent allowed membrane-bound proteins to react completely with the dye reagent. Standard curves of several proteins were similar in the absence or presence of 0.1-0.5% hexyl-beta-D-glucopyranoside. The quantitation of both soluble and membrane-bound proteins by the Bradford assay was similar in the presence of 0.2% hexyl-, heptyl-, and octyl-beta-D-glucopyranoside. Estimates of membrane-bound protein by this assay agreed with estimates obtained with the Lowry assay and with quantitative amino acid analysis. This procedure requires no extra steps; thus, it is as rapid and convenient as the original Bradford protein assay.     

Protein quantification in the presence of poly(ethylene glycol) and dextran using the Bradford method

Some experimental methodologies require the quantification of protein in the presence of polymers like poly(ethylene glycol) (PEG) and dextran (DEX). In the aqueous two-phase system (ATPS) extraction of biomolecules, the interference of these phase-forming polymers on the Bradford quantification assay is commonly recognized. However, how these polymers interfere has not been reported hitherto. In this study we show that while dextran concentrations of 20% (w/w) can be used without error, loss of accuracy occurs for solutions with PEG concentrations >10% (w/w). Above this value a substantial decrease on the assay sensitivity is observed.     

Bradford protein assay and the transition from an insoluble to a soluble dye complex: effects of sodium dodecly sulphate and other additives

The addition of sodium dodecyl sulphate (SDS) (0.0015–0.006%), phenol (0.25–0.5%) or sodium hydroxide (0.025–0.1 M) to the Bradford dye reagent does not improve the solubility of the Coomassie blue-protein dye complex. Centrifugation of the assay tubes, 10 min after the addition of reagent, results in complete loss of colour yield as indicated by the absorbance (A₅₉₅) of the recovered supernates. At protein-concentrations above the working range of tha assay, centrifugation indicates a transition from an insoluble to a soluble protein-dye complex. This transition is characteristic of an individual protein and is influenced by assay modification. Low protein concentrations appear to provide nucleation sites for precipitation of Coomassie blue whilst higher protein concentrations increase its solvation. A soluble dye chromophore is only formed above the working range of the assay indicating that precipitation of the dye protein contributes to the assay mechanism.     

Quantitation of electrophoretic eluted proteins

Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.     

Refinement of the Coomassie brilliant blue G assay for quantitative protein determination

Minor modifications of the Bradford method [1976) Anal. Biochem. 72, 248-254) increase the sensitivity of the Coomassie brilliant blue G microassay for protein quantitation. This is reached by selecting the proper dye source, by alterations in the dye, ethanol, and phosphoric acid concentrations, and by including Triton X-100 in the reaction mixture. The modified assay conditions allow proteins to be detected above 0.2 micrograms and from a solution above 0.4 micrograms/ml. The extinction coefficient of the dye-protein complex was 0.144 +/- 0.010 for 1 microgram bovine serum albumin in the final reaction mixture of 1 ml. In general, the modified procedure exhibits the same specificity, advantages, and drawbacks as described for the original Bradford assay.     

An assay for heparin by decrease in color yield (DECOY) of a protein-dye-binding reaction

The interference by heparin and some related molecules with the well-known Bradford dye-binding assay for proteins is used as the basis of a rapid, sensitive method for the quantitation of these polysaccharides. Whereas the available methods for the assay of glycosaminoglycans have lacked specificity for sulfated polyanions in general and for heparin in particular, the procedure described here distinguishes among different uronic acid derivatives and, when performed in conjunction with heparinase digestion or cetylpyridinium chloride precipitation, can be used to determine heparin content of complex biological samples.     

Interference of sugars in the Coomassie Blue G dye binding assay of proteins

The presence of sugars causes significant deviation from the actual absorbance of proteins in the Bradford protein assay. In these studies, polysaccharides and disaccharides at milligram levels mimicked proteins in microgram equivalents. Monosaccharides, which individually did not show any absorbance, interfered significantly by sequestering the dye species. The studies demonstrated that in a mixture of sugars and proteins, sugar interference was much higher than expected from sugar molecules’ individual contribution. Estimated protein values were increased 2 to 4 times after precipitation from fungal culture broths. Thus, in carbohydrate-rich samples, protein concentrations should be ascertained by precipitation from crude extracts and resolubilization in a noninterfering buffer.     

Protein precipitation of diluted samples in SDS‐containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH‐MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample.     

Protein assay sensitive at nanogram levels

A simple, fast protein assay which utilizes the affinity of colloidal gold for proteins is described. This assay is sensitive at the 20-ng level when a visible light spectrophotometer is used to measure absorbance. Few chemicals interfere with the assay. Interfering reagents include those that are strongly alkaline, contain high levels of salt, or contain sodium dodecyl sulfate. The problem of alkaline interference can be overcome by acidifying the protein solution before performing the assay. Purified proteins have different capacities to interact with the colloidal gold but this variability is not greater than that seen with the Bradford protein assay.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu²⁺) to cuprous ions (Cu¹⁺), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.     

Ecological tannin assays: a critique

Summary The concept of protein precipitation potential has recently been introduced by Wisdom et al. (1987) as a means to combine chemical and protein precipitation assays of tannins for ecological studies. The definition of protein precipitation potential was not theoretically rigorous, and data analysis was obscure. Our attempts to repeat the tannin extraction procedure gave incomplete recovery (24% loss of quebracho) of condensed tannins, the only type considered by Wisdom et al. In contrast we found their method efficient for hydrolysable tannins (80% recovery of tannic acid) which were undetected by their chemical assay of tannins. Their protein precipitation assay was confounded by chemical interference from both types of tannins. We conclude with recommendations for this type of analysis.     

The purification and quantitation of helper T cell-replacing factors secreted by murine spleen cells activated by concanavalin A.

A microculture assay with low numbers of athymic (nude) spleen cells has been used to quantitate the helper T cell-replacing activity secreted by concanavalin A-treated spleen cells. This quantitation allows an estimate of the recovery of biologic activity during concentration from culture supernatants by salt precipitation, and purification by gel filtration, ion exchange chromatography, and isoelectric focusing. The T cell-replacing activity is found in protein of 30 to 40,000 daltons size as estimated by gel filtration, but shows heterogeneity in electric charge. The activity is active at concentrations of less than 10(-9) M, and is strictly antigen dependent in its mode of action.     

A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase

A new and convenient method for the determination of P_i was developed. Phosphomolybdate is measured colorimetrically, without reduction to molybdenum blue, by dissolving the whole assay mixture in acetone, where phosphomolybdate is bright yellow. The hydrolysis of acid-labile phosphates (e.g., creatine phosphate) causes no problems, because extra molybdate is complexed with citrate immediately after the color has been developed. Strong reductants and SH compounds which interfere, if present in high concentrations, are eliminated by adding H₂O₂. Detergents, organic bases, protein, and sucrose do not interfere. The assay is as sensitive as most modifications of the Fiske-SubbaRow method. In the routine procedure the useful range is 50–1500 nmol of P_i. The application of the method to the assay of inorganic pyrophosphatase in the cells of Escherichia coli is described.     

Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates

A novel sensitive binding assay for quantitation of a low-molecular-weight phosphomannosyl receptor (41-46 K) was devised. The receptor was immobilized by immunochemical means in the wells of polystyrene multiwell plates. The lysosomal enzyme ligand, testicular beta-galactosidase, was added and receptor-bound beta-galactosidase was measured by conventional colorimetric analysis using p-nitrophenyl beta-galactoside as substrate. Inhibitors of the binding of beta-galactosidase to the receptor were removed prior to addition of beta-galactosidase and did not interfere with the assay. Low-molecular-weight phosphomannosyl receptor was readily quantitated in the range of 4 to 100 ng of receptor protein. Binding of beta-galactosidase to the receptor was specifically inhibited by 5 mM mannose 6-phosphate. The receptor exhibited optimum binding of beta-galactosidase at pH 5.7 and was saturated with beta-galactosidase at 320 munits/ml in the presence of 20 mM MnCl2. The requirement for MnCl2 was greatly diminished at higher concentrations of beta-galactosidase. Application of the assay procedure to the quantitation of the low-molecular-weight phosphomannosyl receptor in mammalian tissues is discussed.