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 Dynamic Interaction of Coomassie Dye with the Glycine Transporters N-termini

Coomassie Brilliant Blue interacts with proteins and even though the interactions exhibit variation due to the amino acid content, reported dye interactions with individual proteins appear to be relatively stable. Here we report an atypical dynamic interaction of glycine transporters 1 and 2 N-termini with Coomassie dye, resulting in intramolecular interference with their Bradford assay. These proteins exhibit classic protein-Coomassie G-250 complex with absorption maximum at 595 nm, which within minutes starts to decrease and parallel increase of absorbance shoulders above 300 and 700 nm is observed. Interestingly, these effects are eliminated upon fusion of glycine transporters N-termini with glutathione S-transferase protein or by the presence of glutathione S-transferase or bovine serum albumin in the same solution. Circular dichroism data revealed largely unstructured character of glycine transporters N-termini, which suggests that dynamic properties of these protein- Coomassie complexes might be a signature of high flexibility and protein disorder. The assay might potentially reveal similar domains in other proteins and help to associate them with particular functions.     

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.     

Sensitivity and variability of the Bradford protein assay in the presence of detergents

The effects of Triton X-100, sodium dodecyl sulfate (SDS), and urea on the response of Coomassie blue G to 16 different proteins and peptides of Mr 1140 to 146,000 were studied to assess the significance of protein conformation and of ionic and nonionic interactions for the dye response to individual proteins. Triton X-100 at a final concentration of 0.008% (v/v) increased the sensitivity of the Bradford assay toward all proteins of Mr 5700 or higher by an average 33%. Increases ranged from +11% with myelin basic protein to +128% with aprotinin. The relative range of absorbance of proteins and deviations from bovine serum albumin decreased by approximately 25%. Triton X-100 appears to facilitate nonionic interactions of the dye with proteins of limited capacity for ionic binding. Conformation of proteins also seemed to be of some significance because the chaotropic agent urea (0.16 M final concentration) increased sensitivity of the assay by 14%. Sensitivity of the assay was lowered by SDS (0.004% final concentration, w/v) by an average 75% from that of the control assay. The results indicate that the incorporation of low concentrations of a nonionic detergent may be useful in improving sensitivity and variability of the Bradford 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.     

A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue

A method for the quantitation of proteins in solution which involves the binding of bromophenol blue to proteins under acidic conditions has been developed. The binding of the dye to proteins is accompanied by the appearance of a strong absorbance at 610 nm, which is almost linear over the range of 10 to 80 μg for the seven proteins studied. The absorbance at 610 nm can be measured immediately after the mixing of the protein and dye solutions and is stable over a period of 8 hr. The method has very few interferences, most of which can be corrected for by the use of proper controls. Phenol, sodium dodecyl sulfate, and Triton X-100 (the last with some error) may be used with this assay at concentrations that produce strong interferences with similar methods using Coomassie brilliant blue G-250.     

A Coomassie blue-binding assay for the microquantitation of immobilized proteins

A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures.     

Quantitation of proteins using a dye-metal-based colorimetric protein assay

We describe a dye-metal (polyhydroxybenzenesulfonephthalein-type dye and a transition metal) complex-based total protein determination method. The binding of the complex to protein causes a shift in the absorption maximum of the dye-metal complex from 450 to 660 nm. The dye-metal complex has a reddish brown color that changes to green on binding to protein. The color produced from this reaction is stable and increases in a proportional manner over a broad range of protein concentrations. The new Pierce 660 nm Protein Assay is very reproducible, rapid, and more linear compared with the Coomassie dye-based Bradford assay. The assay reagent is room temperature stable, and the assay is a simple and convenient mix-and-read format. The assay has a moderate protein-to-protein variation and is compatible with most detergents, reducing agents, and other commonly used reagents. This is an added advantage for researchers needing to determine protein concentrations in samples containing both detergents and reducing agents.     

Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils

Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for protein concentration determination. In this study, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CB to lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the native protein and to amyloid fibrils but gives distinctly different spectral responses. The absorption and fluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different, and this was most likely due to differences in secondary structure as monitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.     

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.     

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.     

The addition of SDS to the Bradford dye-binding protein assay,a modification with increased sensitivity to collagen

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen in increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result comparable color formation is achieved with both collagens and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 μg. The response to protein is linear up to approximately 40 μg of protein per ml of reagent.     

Quantification of attached cells in tissue culture plates and on microcarriers

A method for quantification of anchorage-dependent cells in culture on plane surfaces or on microcarriers is proposed. It is based on Coomassie brilliant blue R-250 adsorption, followed by elution of the dye and measurement by spectrophotometry at 595 nm. A linear correlation (r = 0.988 to 0.996) was observed between absorbance and cell number along a large range of cell densities. This technique may be used for monitoring cell growth, from seeding of initial inoculi to scaling up of cultures in bioreactors.     

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.     

Interaction of nucleic acids with Coomassie Blue G-250 in the Bradford assay

The Bradford assay has been used reliably for decades to quantify protein in solution. The analyte is incubated in acidic solution of Coomassie Blue G-250 dye, during which reversible ionic and nonionic binding interactions form. Bradford assay color yields were determined for salmon, bovine, shrimp, and kiwi fruit genomic DNA; baker's yeast RNA; bovine serum albumin (BSA); and hen egg lysozyme. Pure DNA and RNA bound the dye, with color yields of 0.0017mg(-1)cm(-1) and 0.0018mg(-1)cm(-1), respectively. The nucleic acid-Coomassie Blue response was significant, at roughly 9% of that for BSA and 18% of that for lysozyme.     

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.     

考马斯亮蓝显色液组分对蛋白质测定的影响

考马斯亮蓝（CBB）显色法测定蛋白质含量的主要缺点之一是线性关系差．通过研究显色液组分对线性关系的影响,发现显色液H⁺浓度是影响线性关系的主要因素,并提出了一个新的显色液配方来改善考马斯亮蓝蛋白质测定法的线性关系．     

A colorimetric assay for measuring the lysis of a plasma clot.

The present study describes a simple, quantitative assay for measuring the lysis of a plasma clot. The principle of the assay is based on the release of Coomassie brilliant blue R-250 dye from the clot. Thirty microliters of freshly prepared Coomassie brilliant blue R-250 (1 mg/ml) was added to 200 microliters of diluted human plasma (1:5). After mixing, 100 microliters of thrombin (2.5 NIH units/ml) were added to mediate a plasma clot. One milliliter of streptokinase (0.1 mg/ml) was used as a plasminogen activator to initiate clot lysis. During the course of lysis, 100 microliters of soluble material were transferred to microtiter wells and the absorbance at 540 nm was determined as a measure of clot lysis. This assay was used to measure clot lysis in 18 human plasma samples. The colorimetric method (X) developed in this report correlated well with that determined using a conventional 125I-fibrinogen method (Y): Y = 0.83X + 7.98 (r = 0.91).     

Linearization of the Bradford Protein Assay

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer''s law perfectly fits the experimental data.Download video file.^{(72M, mp4)}     

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.