Coomassie Brilliant Blue Dye Research Articles

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.

Differences in protein staining by Coomassie Brilliant Blue and neutron activated Coomassie Brilliant Blue dyes

AbstractThe degree of interaction between protein and stains depends in large part upon the protein's content of basic amino acids. We have compared “staining” characteristics of Coomassie Brilliant Blue R‐250 with a dye that was rendered radioactive by neutron bombardment. The protein‐dye intensities were compared to amino acid composition as in our previous study (Electrophoresis 1986, 7, 327–332). While the data for “colored” dye reflected involvement of basic amino acid residues, quite unexpectedly, the radioactive dye appeared to act in part with the acidic amino acids. This may be due to enhanced binding of dye impurities or radiolysis products.

The effects of the availability of diet on the levels of exoglycosidases in the supragingival plaque of macaque monkeys.

Supragingival plaque from macaque monkeys was assayed for 13 exoglycosidase enzymes, with appropriate p-nitrophenylglycosides and N-acetylneuramin-lactose used as substrates. Protein in each plaque sample was quantitated with a Coomassie Brilliant Blue dye binding assay, and the specific activity of each enzyme was calculated. In monkeys fed a starch-based diet, fasting resulted in significant increases in the levels of alpha-L-fucosidase, beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase, and neuraminidase. Such changes are consistent with the hypothesis that plaque bacteria degrade salivary glycoproteins for their growth and maintenance in both the presence and especially the absence of dietary food. In contrast, fasting monkeys previously fed a sucrose-rich diet showed no significant alterations in the specific activities of those enzymes whose levels were increased in the starch-based diet group. It is considered likely that, under the conditions prevailing when the sucrose-rich diet is fed, the effective and maximal utilization of sucrose by plaque bacteria necessitates the increased mobilization of nitrogen sources, including the amino sugars of glycoproteins.

The use of Coomassie brilliant blue for critical micelle concentration determination of detergents

The spectral changes of Coomassie brilliant blue G-250 dye have been studied in the presence of detergent micelles. Association of detergents with the dye produced an absorbance change at 618 nm. At cetyltrimethylammonium bromide concentrations well below the critical micelle concentration a large decrease in absorbance is observed. This is due to formation of a 1:1 water insoluble complex between the anionic dye and the cationic detergent. At sufficiently low dye concentration, the 618-nm absorbance is significantly increased with micelle concentration of the detergent. The onset of enhanced absorbance may be used to determine the critical micelle concentration of cetyltrimethylammonium bromide. The critical micelle concentration of anionic, neutral, and zwitterionic detergents has been determined also by plotting the absorbance at 618 nm as a function of detergent concentration.

Mechanism of dye response and interference in the Bradford protein assay

Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY.

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.

Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis

AbstractA systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R‐250 and G‐250 using a high resolution densitometer allowing for quantitative measurements during staining and destaining has revealed that none of the published procedures allows quantitative measurements. Protein staining with CBB R‐250 in methanol/water/acetic acid is poor, as is staining with CBB G‐250 in trichloroacetic acid or perchloric acid, the latter two, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G‐250 than for R‐250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addition, sensitive protein staining on a clear background is possible. Recipes are described (Section 3.11) for intensified protein staining with CBB G‐250 using trichloroacetic acid or perchloric acid on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G‐250 in the presence of ammonium sulfate since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein‐dye complex. Combining the optimized staining conditions with the stable fixation in 20% ammonium sulfate allows for stepwise staining for e. g. detection of weak spots in addition to intense protein spots. The dependence of different staining procedures on gel thickness, gel concentration and compounds routinely used in polyacrylamide gel electrophoresis is also analysed. Calibration curves and application of the new procedure to biological material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.

Multiple Labeling of Cellular Constituents by Combining Surface Reflection Interference and Fluorescence Microscopy

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.

Fluorography—limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.

An improvement of the Coomassie Blue dye binding method allowing an equal sensitivity to various proteins: application to cerebrospinal fluid

The Coomassie Brilliant Blue dye binding method for protein determination is of limited value due to an unequal sensitivity to various proteins. Addition of sodium dodecyl sulfate to the reagent equalizes the reactivity for human albumin, transferrin, IgG, thus making its use for cerebrospinal fluid protein assay valid. Both a manual and an automated method have been described. A good correlation existed: the former with the method of Meulemans ( r = 0.996), the latter with the method of Lowry ( r = 0.987).

Particulate protein measurement in oceanographic samples by dye binding

A modified version of the Coomassie Brilliant Blue dye binding protein assay has been developed for oceanographic samples and intercalibrated with the widely used Lowry assay. Particulate protein measurements were made at seven stations in the Gulf of Maine using the method. Measurements were made on cell-free homogenates. Protein concentration ranged from 2 to 212 μg l −1 (0.02–0.68 μg at N l −1) and averaged 58 μg l −1. ETS activity, chlorophyll and particulate nitrogen were significantly correlated with protein concentrations.

Rapid, automated measurements of urinary protein and glucose concentrations

Procedures are described for the quantitative determination of urinary protein and glucose concentrations using assay methodologies that are readily automated. Glucose was measured by the hexokinase method and protein by interaction with the dye Coomassie Brilliant Blue C-250. Both assays can be performed with a centrifugal analyzer. The linear range for the glucose assay was 10–500 mg dl , encompassing the normal range of 10–30 mg dl for glucose in rat urine. The linear range for the protein assay was 5–45 mg dl , necessitating dilution of the urine (normal range for rats of 100–400 mg dl ) by a factor of 10. As with many other protein assay methodologies, response to gamma globulins was less than that to albumin. The procedures described herein require only minute quantities (5–10 μl) of sample and are sufficiently simple and rapid to be used as screening procedures. Moreover, they provide quantitative results and are more sensitive than many of the more commonly used diagnostic tests for urine protein and glucose.

Automated determination of urine and cerebrospinal fluid proteins with coomassie brilliant blue and the abbott ABA-100

We have evaluated the use of Coomassie Brilliant Blue (CBB) dye to measure the protein of urine and cerebral spinal fluid. The method was automated and compared with the turbidimetric method and with Ponceau S dye. With both the manual and automated procedures the CBB method could be used to protein concentrations of 160 mg/dl. Comparing this method to the method measuring the turbidity produced by trichloracetic acid (TCA) we found a correlation coefficient (r) of 0.96. The regression line was CBB = 0.84 TCA + 4.97. A similar comparison to the method using Ponceau S (PS) produced an r = 0.98 and the regression line, CBB = 0.91 P-S + 3.2. The CBB method was not as affected by turbidity in the samples or by xanthochromia as was the TCA Method. It is simpler to perform than the P-S method, since it does not require decantation. With the manual method, the coefficient of variation (CV) at 40 mg/dl for within-day determinations was less than 2% and, for between-day determinations, less than 4%. While the CV was somewhat higher with the ABA than with the manual method at 40 mg/dl, it was below 5% for both within-day and between-day determinations at the concentrations between 70 and 170 mg/dl. The method using CBB is useful as both a manual and automated procedure to measure protein in urine and cerebral spinal fluid.