The use of high-pressure liquid chromatography in the simultaneous assay of 11beta-hydroxylase and 18-hydroxylase in zona fasciculata-reticularis tissue of the rat adrenal cortex.

Certain reagents utilized in the formation of polyacrylamide gels are shown to interfere in the Lowry assay for protein. Acrylamide (3–30%) and potassium ferrocyanide (0.0015–0.0105%) produced a linear response in color formation. Both compounds are capable of reducing the phenol reagent in the absence of copper and the interference can be compensated for by employing the appropriate blank. An extract of polymerized and electrophoresed gels also interferes in the Lowry assay, however, this increased color formation cannot be corrected by using a gel extract blank. Under the conditions studied, filtration, centrifugation, and dialysis did not sufficiently remove the acrylamide fines responsible for the interference.     

Comparative study of Lowry and Bradford methods: interfering substances

The methods of Lowry and Bradford, commonly used for protein determination, were compared regarding the level of interference of some substances used for glucoamylase precipitation by ethanol. The method of Bradford suffers no interference while the method of Lowry showed protein concentration values 20% increased in the presence of ethanol and Tris. Despite these interferences, the Lowry method can evaluate more accurately the increase of purity during fractionation, due to its sensitivity to low molecular weight (below 6 kDa) proteins and peptides.     

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.     

A tracking marker for the first dimension of the two-dimensional gel electrophoresis of ribosomal proteins

Protein content of different subcellular fractions from chick brain is compared by using Lowry, TCA—Lowry and Bradford assay methods. Caution is urged in application of Bradford's method to general assay for protein concentration in subcellular fractions.     

Interference by Tris buffer in the estimation of protein by the Lowry procedure

Tris buffers were found to distort the measurement of protein by the Lowry method both by decreasing chromophore development with protein and by contributing blank color. Tris at an assay concentration of 0.37 mm markedly affects measured results. Similar Tris effects were observed at all wavelengths between 450 and 800 nm and with diverse protein samples. The distortion due to Tris is not correctable by simple blank correction, but it can be overcome by incorporating the same amount of Tris in the standards used. The distortion at Tris concentrations <0.15 mm appears to be within tolerable limits. No interference or distortion was observed with sodium phosphate buffer to an assay concentration of 40 mm. An automated Lowry procedure is also presented which gives excellent correlation with the manual method and an average coefficient of variation of <4%.     

Lowry method of protein quantification: Evidence for photosensitivity

Despite reports of its susceptibility to various interfering factors, the Folin Phenol protein quantification method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265–275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken.     

Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80

Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances.     

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.     

A simplification of the protein assay method of Lowry et al. which is more generally applicable

Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.     

重组人白介素-11蛋白浓度两种测定方法比较

在基因工程蛋白药物开发研究过程中,选定恰当、准确的蛋白浓度测定方法非常重要。本文比较Lowry法和Bradford法测定重组人白介素-11蛋白浓度结果,认为选用Bradford法是可行的,但中国药典三部蛋白浓度测定方法中没有收录Bradford法,建议在中国药典三部修订时收录该方法。     

Measurement of total protein in plant samples in the presence of tannins

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.     

Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.     

A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates

Summary Clostridium thermocellum culture media containing crude cellulosic substrates, prepared by the steam-explosion of wood or straw, were found to interfere strongly with the Lowry method of protein determination. Investigations into the mode of interferences demonstrated that either the intercept (blank value) and/or the slope (intensity of color development) were affected. The efficiencies of precipitation procedures using the trichloroacetic acid method and the deoxycholate-trichloroacetic acid method were compared.     

Problems associated with determining protein concentration: a comparison of techniques for protein estimations

Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric analysis and densitometry of stained protein gels, are applied, but perhaps to a lesser extent. However, the reliability of all of the above techniques is questionable and dependent to some extent on the protein to be assayed. In this paper we describe problems we encountered when using some of the foregoing techniques to quantify the concentration of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), a nuclear enzyme found in most eukaryotes. We also describe how, by using a fluorescence-based assay and amino acid analysis, we overcame the problems we encountered.     

Influence of carbamoylation on some analytical properties of basic polypeptides

The effect of carbamoylation on the assay or identification of histones and polylysine was investigated. Incubation with sodium cyanate decreased the positive charge on these polypeptides as judged by changes in the binding of methyl orange or the electrophoretic mobility. Histones in chromatin appeared less accessible to carbamoylation than isolated histones. Carbamoylation of proteins under conditions in which there was little or no effect on the Lowry procedure could affect their assay by methods utilizing metachromasia with Coomassie Blue G. The Bradford assay has low sensitivity for Hl histone and polylysine but this can be increased by preincubation with sodium cyanate. More extensive carbamoylation of polylysine caused decreased sensitivity which was the only response seen with core nucleosomal histones and bovine serum albumin when preincubated with sodium cyanate. It was concluded that the sensitivity for Hl histone and polylysine in assays dependent on metachromasia with Coomassie Blue G may be changed by factors which decrease the positive charge on these polypeptides.     

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.     

A procedure for eliminating interferences in the lowry method of protein determination

A modification of the Lowry assay for the quantitative protein measurement in the presence of interfering materials has been developed. The method is based on a precipitation with a single-phase hexane:isopropanol solvent system and later resuspension of protein pellets with sodium dodecyl sulfate and deoxycholate. The new procedure eliminates the interference caused by Triton X-100, phospholipids, or dithiothreitol providing yields higher than 95% and seems to be especially suitable for protein determination on membrane preparations in samples with small volumes and/or very low protein concentrations.     

Fiftyfold amplification of the Lowry protein assay

The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenol addition to the Bradford dye binding assay improves sensitivity and gives a characteristic response with different proteins

Phenol has been added to the Coomassie Brilliant Blue G-250 dye reagent used in the standard Bradford protein assay and its effect upon the reagent blank and assay response of fourteen proteins investigated. Phenol can enhance or impair colour yield depending upon its concentration and the amount and type of protein assayed. Four characteristic protein responses to increasing assay concentrations of phenol have been observed. These indicate a complex influence of phenol upon the protein assay. Dye reagent containing 0.5% phenol gave optimal colour yield with most of the proteins investigated and an improved assay response of ovalbumin, ribonuclease, lysozyme, insulin, pepsin and chymotrypsinogen-A relative to bovine albumin.     

Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method

Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process.