An Update on Protein Stains: Amido Black, Coomassie Blue G, and Coomassie Blue R

Samples of amido black, Coomassie blue G, and Coomassie blue R obtained over a number of years were tested for dye content, impurities, and effectiveness for staining proteins after polyacrylamide gel electrophoresis and for protein dye-binding assays. Some impurities produced reactions resembling metachromasia with specific proteins, although instances of true metachromatic staining are also reported. Several simple assays are given for determining dye content and relative levels of impurities. Recommendations are made for selecting batches of commercial dyes which are most likely to perform satisfactorily.     

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis

Blue native gel electrophoresis (BN–PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN–PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN–PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed.     

Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis.

The properties of amido black 10B (C.I. 20470), Coomassie blue R (C.I. 42660), and fast green FCF (C.I. 42053) as protein stains, along with a few comments on Coomassie blue G (C.I. 42655), are presented and dye impurities and their effects on protein-dye binding within gels are discussed. All three dyes produced metachromatic effects with some proteins. Problems encountered with long-term stability and fixation of certain maize seed proteins are reported along with procedures for overcoming them. The low solubility of Coomassie blue R in trichloroacetic acid prevented maximum staining and destaining within a reasonable time, whereas other solvents allowed diffusion of some proteins during staining. Coomassie blue R binds to proteins in much higher amounts than do amido black and fast green, which accounts for its sensitivity in detection of protein bands in gels. Procedures for obtaining maximum contrast with photographs are also outlined.     

Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

A method to quantitate Coomassie blue-stained proteins in cylindrical polyacrylamide gels

A method for the quantitation of Coomassie blue-stained proteins in cylindrical polyacrylamide gels is described. It involves an elution of the dye with an 80% methanol solution in a sealed Pyrex tube at 100 degrees C for 3 h and a measurement of its concentration at 585 nm. Using a 6.5% polyacrylamide gel and bovine serum albumin as a protein standard, the curve of absorbance of the dye solution as a function of the amount of protein was observed to be linear up to 30-40 micrograms of protein and as little as 0.8-1.0 micrograms of protein could be measured. The validity of the method was indicated by the values obtained for the relative proportions of the human erythrocyte membrane proteins. Using this method, the color yields of several proteins varying widely with respect to their size, amino acid composition, and carbohydrate content were determined in a 6.5% polyacrylamide gel. The results showed that they were generally the same except for proteins having a high carbohydrate content which were significantly lower.     

Alcian Blue Pyridine Variant-a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

低毒高效的SDS-PAGE考马斯亮蓝染色方法比较

目的:比较SDS-PAGE的4种考马斯亮蓝染色方法。方法:以牛血清白蛋白为材料进行SDS-PAGE,分别比较考马斯亮蓝G-250(CBB G-250)盐酸法、CBB G-250-Al2(SO4)3法、Bio-Rad公司的Bio-Safe染色液及传统法等4种染色方法的灵敏度和操作性,并将上述4种染色方法应用于蛋白Markers检测。结果:CBB G-250-Al2(SO4)3法和Bio-Safe法的检测灵敏度都可达19.2 ng,而CBB G-250盐酸法和传统法的检测灵敏度则为28.9 ng。结论:CBB G-250盐酸法可作为快速、低毒、高效的染色方法,CBB-Al2(SO4)3法则可用于灵敏度要求较高的检测。     

Enhanced thiosulfate-silver staining of proteins in gels using Coomassie Blue and chromium

Potassium chromium sulfate, a new sensitivity enhancer for silver staining of proteins in gels, enhanced the sensitivity of the thiosulfate-silver staining method. The sensitivity could be further improved when potassium chromium sulfate was used in association with another sensitivity enhancer, Coomassie Brilliant Blue R-250 (CBB R-250). The sensitivity of the CBB-chromium modified method to strongly basic proteins such as ribosomal proteins was about 20-fold over that of the published method. This novel method has direct applicability for 2-D gel electrophoresis used in proteomics.     

Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.     

Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein

A method has been developed to determine the relative or actual number of attached cells in microtiter plate wells without making direct cell counts. The procedure is based upon staining total cellular protein with Coomassie brilliant blue G-250, followed by measurement of absorbance at 630 nm in a spectrophotometer designed to read each well of a 96-well microtiter plate. No destaining of cells is required. A linear correlation exists (r = 0.970) between cell number and absorbance over a useful range. Intraplate well-to-well variation is acceptable (CV = 0.101). This method was used to measure the proliferative response of human vascular smooth muscle cells to human serum. It should be useful in other assays involving proliferation of attached cultured cells.     

考马斯亮蓝与牛血清白蛋白相互作用机理的研究

利用光谱探针技术研究在酸性溶液中考马斯亮蓝G-250(Coomassie brilliant blue G-250,CBBG)与牛血清白蛋白(bovine serum albumin,BSA)相互作用机理,考察了不同实验条件对CBBG-BSA复合物吸收光谱的影响。实验结果表明:CBBG与BSA相互作用产生光谱蓝移主要是由CBBG与BSA间的疏水相互作用引起,而静电作用则是形成CBBG-BSA蓝色复合物的必要条件。同时,CBBG聚集体的聚集程度是影响CBBG-BSA蓝色复合物形成的重要因素。     

Demonstration of Axial Elements of Sex Vesicles of Spermatocytes Using Coomassie Brilliant Blue

The axial element of sex chromosomes in the sex vesicle of rat and mouse spermatocytes has been visualized under the light microscope by the dye Coomassie brilliant blue (CBB). After staining in the CBB solution for 3-10 minutes, the axial elements appeared as darkly stained threads in the sex vesicles, whereas in controls stained with Giemsa or carbol fuchsin, the sex vesicles were usually uniformly stained. The axial elements are best seen when chromosome preparations were made by the flame drying technique. In rat spermatocytes the staining quality could be further improved by a brief treatment with trypsin solution (0.025%).

The CBB staining procedure is simple and easily controllable. The results suggest that the CBB stained material is protein in nature and is more resistant to trypsin digestion than other nuclear proteins.     

A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay

A simple procedure for the determination of cellular proteins in Percoll-containing samples is described. Percoll precipitated when particulate proteins were solubilized by dilution of the samples in a NaOH-Triton X-100 mixture. After centrifugation at high speed (12,000g), the supernatant was assayed for proteins with the Coomassie brilliant blue dye-binding assay. With an automatic spectrophotometer, 50-microliter aliquots gave a linear response between 0 and 3 micrograms of bovine serum albumin. After a fivefold dilution in the alkali-detergent mixture, proteins in samples containing up to at least 60% Percoll can be accurately quantitated on a standard curve prepared in the absence of Percoll. Because the sensitivity of the assay was better than 100 ng, the procedure outlined in this paper can also be used as a general protein micromethod.     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Determination of protein by Coomassie dye-binding in agarose gels

A variation of the Coomassie dye-binding assay for proteins is described. Protein samples were pipetted to the surface of agarose plates in uniformly sized spots and stained with Coomassie Blue G-250. The bound dye was determined by densitometric scanning using double wavelength and flying spot facilities. The response curves were linear in an about 10-fold concentration range with a lower detection limit of 0.5 microgram. No background correction was necessary because unbound dye and most substances known to interfere with other protein assays were removed during the staining and destaining of the agarose gels. Membrane proteins could be analyzed since the samples were applied as solutions in 1% sodium dodecyl sulfate.     

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.     

A modified Coomassie Brilliant Blue staining method at nanogram sensitivity compatible with proteomic analysis

A more sensitive and convenient Coomassie Brilliant Blue (CBB) staining method for visualizing proteins was developed. Compared with the modifications include the supplement of 10% (v/v) methanol into the fixing solution, an increase of an additional sensitization step and CBB raised from 0.1 to 0.125%. The improved method can detect proteins at nanogram level. The improved method is more sensitive than Blue Silver and more convenient than the Silver protocol. Mass spectrometry results confirmed that it is suitable for subsequent proteomic research.     

Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain

Gel destaining following Coomassie Brilliant Blue (CBB) staining involves the use of toxic reagents. Here we demonstrate the efficacy of various paper adsorbents in adsorbing CBB. Kimwipes adsorbed the best, followed by Teri towels, multifold towels, and Whatman numbers 1 and 3 filter papers. Three Kimwipes completely adsorbed the dye released from a CBB-stained mini-gel. Nonradioactive destain solution can, therefore, be recycled for destaining CBB-stained gels. Stain removal with Kimwipes helps in reducing destain use and in reducing organic liquid waste, and it is 7.5-fold cheaper compared with an available method for CBB disposal. Following this, we determined the suitability of this procedure to remove the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. The dye from a 0.05% CBB staining solution could be removed in 5 to 10 min using 75 Kimwipes. The CBB-adsorbed Kimwipes did not release the stain when squeezed dry even after incubation in various salts over 1 week and in water for 5 weeks. The CBB removed allows its easy disposal as solid waste and will not leach out from solid landfills. Thus, stain removal with Kimwipes helps in disposing CBB in an environmentally friendly manner and allows recycling of destaining solution.