Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins

We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays.     

Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie brilliant blue

Low enzyme concentrations can be determined quantitatively using rocket immunoelectrophoresis if the resolved peaks are transferred electrophoretically to a nitrocellulose membrane and immunostained.     

Application of Neuhoff's optimized Coomassie brilliant blue G-250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.     

Interference of the carbohydrate moiety in coomassie brilliant blue R-250 protein staining

The binding of Coomassie Brilliant Blue R-250 to several species of bovine pancreatic ribonuclease is affected by the presence of a carbohydrate moiety in the enzyme molecule. Enzymic deglycosylation of several chromatographic fractions of ribonuclease, which have different degrees of glycosylation, results in increased staining by Coomassie Brilliant Blue R-250. Ovalbumin and other glycoproteins tested show similar behavior. The results indicate that carbohydrate moieties may represent a common hindrance to the binding of Coomassie Brilliant Blue dyes to glycoproteins.     

饮料及糖果中亮蓝、苋菜红及柠檬黄的简便定量分析EI北大核心CSCD

采用定性与定量相结合,建立了测定饮料及糖果中共存色素亮蓝(BRIB)、苋菜红(AMA)及柠檬黄(TAR)的简便吸收光谱法。BRIB,AMA及TAR在pH 3.50 Tris-HCl条件下,分别与乙基绿反应生成离子缔合物,产生新的特征吸收光谱。定量测定波长分别为588 nm(BRIB体系)、532 nm(AMA体系)和426 nm(TAR体系),表观摩尔吸光系数(κ)分别为4.24×10^(4)L/(mol·cm)(BRIB体系)、3.35×10^(4)L/(mol·cm)(AMA体系)和2.64×10^(4)L/(mol·cm)(TAR体系),线性范围分别为0.05~18.8 mg/L(BRIB体系)、0.05~13.3 mg/L(AMA体系)和0.05~11.7 mg/L(TAR体系),检出限分别为35μg/L(BRIB体系)、27μg/L(AMA体系)和30μg/L(TAR体系),定量限分别为0.61 mg/kg(BRIB体系)、0.47 mg/kg(AMA体系)和0.52 mg/kg(TAR体系)。该方法用于市售饮料及糖果中共存BRIB,AMA及TAR的测定,加标回收率为97.8%~103%(BRIB体系)、97.9%~102%(AMA体系)和98.1%~103%(TAR体系),相对标准偏差分别为2.0%~2.8%(BRIB体系),1.2%~2.6%(AMA体系)和1.6%~2.6%(TAR体系)。     

Recovery of protein by coomassie brilliant blue precipitation prior to electrophoresis.

The interaction of protein with Coomassie Brilliant Blue G-250 results in formation of an insoluble protein-dye complex which can be recovered by centrifugation and redissolved for electrophoretic analysis. The precipitated protein can be washed in acetone to remove excess dye in order to enhance resolution. The residual dye becomes dissociated from the proteins on electrophoresis and can be exploited as a "dye front". The method allows simultaneous protein assay and recovery of microgram amounts of protein from dilute solution and could be widely applied for conserving, concentrating and desalting minute amounts of valuable sample prior to electrophoretic analysis.     

Molecular spectroscopy study of the reaction of nucleic acids with brilliant cresol blue

The interaction of brilliant cresol blue (BCB) with nucleic acids in aqueous solution has been studied by spectrophotometry and Rayleigh light scattering (RLS) spectroscopy. Under suitable conditions, the RLS spectra of BCB changed significantly due to the presence of nucleic acids. RLS intensity of BCB at 364 nm is greatly enhanced with the addition of nucleic acids, and a new RLS peak is observed at 552 nm. This peak is about half the intensity of that at 364 nm. The results of this study show that BCB interacts with DNA possibly due to the cooperative effect of electrostatic attraction, intercalation, coordination and hydrophobic effect. Under optimum conditions, the increase of RLS at 364 nm of a BCB solution is proportional to the concentration of nucleic acids added. This result is the basis for a new RLS method for determination of nucleic acids. The linear range of ctDNA, fsDNA and yRNA is 0.12-4.70, 0.11-4.64 and 0.43-7.07 microg ml(-1), respectively.     

Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate.

Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.     

考马斯亮蓝G光度法测定钼的研究

考马斯亮蓝Ｇ是一种生物染色剂 ,在分析测定中多用于蛋白质的测定[1 ,2 ] 。近年也有人用此试剂做催化动力学分析中的指示剂[3 ,4] 。我们在研究中发现 ,考马斯亮蓝Ｇ用于缔合物体系测定阳离子 ,有较好的结果。本文研究了以铜离子为催化剂 ,在抗坏血酸存在下的钼 硫氰酸盐 考马斯亮蓝显色体系 ,建立了一个灵敏的测定钼的方法。该方法灵敏度高 ,其表观摩尔吸光系数ε50 0 =2 .3 1×1 0 5Ｌ·ｍｏｌ- 1 ·ｃｍ- 1 。线性范围宽 ,钼的浓度在 0～2 1 μｇ/2 5ｍＬ范围内符合比尔定律。选择性较高 ,显色时间短 ,适合于生产中使用。该方法可用于合…     

A single step protein assay that is both detergent and reducer compatible: The cydex blue assay

Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue‐based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single‐step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta‐cyclodextrin or alpha‐cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2–10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay.     

蛋白质与酸性铬蓝K相互作用的分光光度研究

本文研究了不同酸度下, 酸性铬蓝K与牛血清白蛋白的作用情况。用光度法求出了不同条件下酸性铬蓝K与牛血清白蛋白的结合个数或结合常数, 并用三种不同的方法相互进行对照。证明酸性铬蓝K在牛血清白蛋白上有两类不同结合部位。     

Complexation study of brilliant cresyl blue with beta-cyclodextrin and its derivatives by UV-vis and fluorospectrometry

The complexation reactions of brilliant cresyl blue (BCB) with beta-cyclodextrin (beta-CD), mono[2-O-(2-hydroxypropyl)]-beta-CD (2-HP-beta-CD), mono[2-O-(2-hydroxyethyl)]-beta-CD (2-HE-beta-CD), and heptakis(2,6-di-methyl) -beta-CD (DM-beta-CD) were investigated using UV-vis and fluorospectrometry. The complexation between BCB and CDs could inhibit the aggregation of BCB molecules and could cause its absorbance at 634nm gradually increasing. The fluorescence of BCB was also enhanced with the addition of CDs. The fluorescence enhancement was more notable in neutral and acidic media than in basic media. Hildebrand-Benesi equation was used to calculate the formation constants of beta-CDs with BCB based on the fluorescence differences in the CDs solution. The stoichiometry ratio was found to be 1:1. The complexing capacities of beta-CD and its three derivatives were compared and the results followed the order: 2-HP-beta-CD>2-HE-beta-CD>DM-beta-CD>beta-CD. The effect of temperature on the formation of BCB-beta-CD inclusion complexes has also been examined. The results revealed that the formation constants decreased with the increase of temperature from 1038.9 to 491.6l/mol. Enthalpy and entropy values were calculated and the values were -25.77kJ/mol and 35.04J/kmol, respectively. The thermodynamic measurements suggest that the inclusive process was enthalpic favor. The release of high-energy water molecules and Van der Waals force played an important role in the inclusive process.     

Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.     

A new method of estimating monomer reactivity ratios in copolymerization by linear regression methods

Sequential gas-liquid chromotographic analysis of the reaction mixture throughout a copolymerization reaction in conjuction with the improved curve-fitting I (integrated form) method, which accounts for measurements errors in both variables, allows accurate estimation of the monomer reactivity ratios. In this article an alternative method is presented for estimating r values in copolymerization with linear regression only, which is especially suited to cases in which one or two of the r values is close to 1. In these cases the improved curve-fitting I method tends to converge slowly because of the numerical instability of the integrated copolymerization equation. The use of the new method is illustrated for the estimation of the r values for ethylene and vinyl acatate in benzene at 35 kg/cm² and 62°C. The linear regression method was also tried on other copolymerizations and the results are compared with those obtained from the improved curve-fitting I method. The limits of the applicability of the linear regression method were determined by simulated sequential sampling experiments. It appears that the new method is applicable when the product of the r values is between 0.001 and 2, provided both monomer conversions are large enough compared with the measurements error.     

Interaction of brilliant red X-3B with bovine serum albumin and application to protein assay

The interaction of brilliant red X-3B (BRX) with bovine serum albumin (BSA) in three pH media has been characterized by the spectral correction technique. The binding number maximum of BRX was determined to be 102 at pH 2.03, 82 at pH 3.25 and 38 at pH 4.35 and the binding mechanism was analyzed in detail. The effects of ionic strength from 0 to 1 mol L⁻¹ and temperature from 20 to 70 °C on the binding were investigated. The results showed that the interaction of BRX with BSA responded to the Langmuir adsorption isothermal model and the binding constant was determined. From the correlation between the binding number and the number of basic amino acid residues, the ion-pair attraction induced the union of non-covalent bonds including H-bond, van der Waals force and hydrophobic bond and the binding model was illustrated. The binding of BRX to BSA has resulted in change of the BSA conformation confirmed by means of circular dichroism. Using this interaction at pH 2.03, a sensitive method named the absorbance ratio difference spectrometry was established and applied to the protein assay and the limit of detection of protein was only 6 μg L⁻¹. Two samples were determined and the results were in agreement with those obtained by the classical coomassie brilliant blue colorimetry.     

Comparison of regression techniques for linear calibration

A recently presented regression technique for linear calibration, which is based on a variance component model for univariate quantitative measurement data, is compared with the conventional and far spread regression techniques ordinary least squares regression and weighted least squares regression. The associated statistical models and estimations are represented. Its application is demonstrated at some practical examples. With consideration of special variation causes, like matrix influence or the influence of several operating conditions on the measurement response, it can be shown that the application of the variance component model is an advantage.     

A colorimetric method for the assay of lipoyl dehydrogenase

Microchimica Acta - A colorimetric method for the direct assay of lipoyl dehydrogenase is described. Enzyme reaction is stopped by ethanol precipitation. This is followed by the displacement of...     

A method for enlarging the Kerr constant of polymer-stabilised blue phases

Polymer-stabilised blue phase (PSBP) is one of the most promising materials for display devices because of its superior electro-optical properties compared with conventional nematics. However, the application of the PSBP has a serious practical issue in that the driving voltage required is too high to drive with thin film transitors, that is, the magnitude of the Kerr constant of PSBPs is insufficient. We present a useful method for increasing the Kerr constant based on the control of polymer aggregation structure using a cross-linker with a chiral structure. The director distortion arising from polymer networks in the PSBP seems to be responsible for the resulting improvement.     

The brilliant blue FCF ion-molecular forms in solutions according to the spectrophotometry data

The brilliant blue FCF acid–base properties in aqueous solutions have been studied and its ionization constants have been defined by tristimulus colorimetry and spectrophotometry methods. The scheme of the acid–base dye equilibrium has been proposed and a diagram of the distribution of its ionic-molecular forms has been built. It has been established that the dominant form of the dye was the electroneutral form, which molar absorptivity (ε₆₂₅ = 0.97 × 10⁵) increases with the increase of the dielectric permittivity of the solvent. It has been shown that the replacement of polar solvents by less polar ones is causing a bathochromic shift of the maximum absorption band of the dye, the value of which is correlated with the value of the Hansen parameter. Tautomerization constants have been defined in a number of solvents and associated with the value of the Dimroth-Reichardt parameter.