Isolation of a component from commercial coomassie brilliant blue R-250 that stains rubrophilin and other proteins red on polyacrylamide gels

Commercially available Coomassie Brilliant Blue R-250 (C.I. 42660) is a popular and useful dye that stains most proteins blue on polyacrylamide gels. Some proteins from brain (rubrophilin), collagens, histones and parotid gland proteins are distinctly red when stained with Coomassie Blue. Commonly used Coomassie Brilliant Blue R-250 preparations may contain more than 30 distinct colored and fluorescent components that can be separated on silica gel chromatographic columns. A specific component has been isolated on silica gel columns that stains rubrophilin and other proline-rich proteins a reddish color. Fast atom bombardment mass spectrometry of the isolated rubrophilin staining principle indicates a molecular weight of 634 as compared to 826 for the major dye in the original Coomassie Brilliant Blue R-250. Infrared spectrometry is consistent with a difference between the rubrophilin staining principle and Coomassie Brilliant Blue R-250 of a toluene sulfonic acid residue.     

Direct red 81 and amido black stain proteins in polyacrylamide electrophoresis gels within 10 min

Proteins separated by SDS-polyacrylamide gel electrophoresis can be stained with organic dyes, the most popular being Coomassie brilliant blue R-250. Coomassie R-250 staining of ovalbumin in an SDS-PAGE gel increased linearly from 2.5 to 60 min. Direct red 81 and amido black staining approached saturation in 10 min. Scatchard analysis showed that the number of direct red 81 and amido black ligands bound to ovalbumin was fourfold higher than that of Coomassie R-250. Direct red 81 and amido black stain proteins in an SDS-polyacrylamide electrophoresis gel in 10 min.     

Specific indication of hemoproteins in polyacrylamide gels using a double-staining process

Hemoproteins were revealed in polyacrylamide gels in the presence of sodium dodecyl sulfate by staining with different benzidine derivatives. When the protein samples were treated with either beta-mercaptoethanol or dithiothreitol, a significant decrease in peroxidase activity of the proteins possessing noncovalently bound heme led to diminished staining. However, when Coomassie blue R-250 staining followed the hemespecific stain it was observed that the hemoprotein bands stained more intensely than duplicate sample bands that had been stained only with the Coomassie blue R-250. This staining property allows the indication of hemoproteins in gels even after the peroxidase yield has been significantly depleted by reducing agents.     

Advantages and disadvantages of silver staining of proteins in polyacrylamide gel

Sensitivity of protein staining with Serva blue G-250 (Coomassie brilliant blue G-250 analogue) in polyacrylamide gel was determined. It has been shown that protein staining with 0.1% Serva blue G-250 results in the recovery of 80 to 35 ng of single protein, that is almost 10 times higher than reported previously for Coomassie brill. blue G-250 (or R-250) staining. The comparison of the sensitivity of Serva blue G-250 protein staining in PAAG and AgNO3 has shown that AgNO3 staining was approximately 18-30 (but not 100 times, as it had been thought before) times more effective for the majority of proteins under study. Silver staining of some proteins, for instance ribonuclease and a number of retrovirus-specific structural proteins, was of lower efficacy. Thus, to obtain reliable results protein electrophoresis in PAAG should be followed by both staining procedures.     

荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色效果比较*

比较了荧光素钠和考马斯亮蓝应用于小麦白粉病菌染色的效果。荧光素钠法中样品处理只需20min.左右,具有直接、快速的特点;荧光指示剂对病菌分生孢子萌发及菌丝生长无抑制作用,主要沉集于活菌体的隔膜和细胞质部位,使病菌产生明显的亮绿荧光和清晰的细胞轮廓,亮绿荧光衰退期为7min.;借助荧光显微镜可以观察病菌在小麦叶表的发展过程,区别活菌体和失活菌体。考马斯亮蓝法包括传统的组织学染色步骤,经过改进后的样品处理过程需要40min.左右;染色后使寄主组织呈现淡蓝色,病菌菌体染成深蓝色;该方法可以观察病菌在小麦叶表和被侵染细胞内部发育形成的结构,包括孢子发育形成的初生芽管、附着胞芽管、成熟附着胞以及在寄主细胞内形成的初生吸器原体、成熟的指状体吸器和次生吸器。     

A fast silver staining method for protein detection after isoelectric focusing in agarose gels

A sensitive staining method was developed for detecting proteins in agarose gels after isoelectric focusing. Its sensitivity is about 20 times that of the Coomassie blue R-250 staining technique, and the time required is only 10 min.     

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.     

Staining protein in isoelectric focusing gels with fast green

A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated.     

Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w/v, in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng.microliter-1, respectively.     

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).     

Immunodetection after complete destaining of coomassie blue-stained proteins on immobilon-PVDF.

A technique that simplifies the localization of an immunodetectable protein in relation to the other electrophoresed proteins is described. Proteins are transblotted onto a polyvinylidene difluoride (PVDF) membrane and visualized by staining with Coomassie brilliant blue R-250, and a photograph of the protein pattern is taken. The Coomassie blue-stained PVDF membrane is then completely destained using a 25% acetic acid/50% methanol solution that allows subsequent immunostaining on the same membrane. The technique uses common laboratory reagents, is rapid, and has been shown to be applicable for a variety of proteins using both monoclonal and polyclonal antibodies and a variety of transblots.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.     

Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel

The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.     

Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer

Dimethyl sulfoxide was found to be effective for extraction of Coomassie Brilliant Blue R-250 (Coomassie R) from stained proteins on polyacrylamide gel slices. A good correlation was found between the ability of different proteins to bind Coomassie R and their capacity for interaction with Coomassie Brilliant Blue G-250 (Coomassie G) in solution. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge.     

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.     

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.     

Decreased Coomassie brilliant blue colour yield with glycated proteins.

Collagen, myosin and albumin were incubated for 7 days at 20 degrees C with fructose, ribose or glyceraldehyde. For thus-formed glycated proteins, quantities were determined by the Conway microdiffusion technique and by the colorimetric method based on Coomassie brilliant blue G-250 colour yield. It was found that when albumin was glycated with increasing amounts of glyceraldehyde, the colour yield was decreased by 7-33%. In collagen, myosin and albumin incubated with 0.5 mol/l fructose, 0.5 mol/l ribose or 0.1 mol/l glyceraldehyde, protein concentration was not changed, as proved by the Conway microdiffusion technique; the Coomassie brilliant blue G-250 colour yield was up to 50% lower, depending on the protein used, and was decreased much less when proteins were incubated with less sugar.     

肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用

目的：研究肌肽对低氧所致大鼠血管内皮细胞损伤的影响。方法：建立低氧条件下大鼠血管内皮细胞损伤模型,用MTT法观察肌肽对低氧损伤的血管内皮细胞活性的影响,测定细胞培养基中LDH活力,并对细胞骨架进行考马斯亮蓝R-250染色观测其细胞结构。结果：浓度为10mmol/L～20mmol/L肌肽孵育血管内皮细胞6h后,可以抑制缺氧12h和24h引起的血管内皮细胞活性下降,同时减少LDH的释放,保持细胞骨架完整。结论：肌肽对低氧所致的血管内皮细胞损伤具有保护作用。     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Quantitation of types I and III collagens in human tissue samples and cell culture by cyanogen bromide peptide analysis

A procedure for the quantitation of types I and III collagens by cyanogen bromide peptide analysis was developed with the aim of eliminating certain problems associated with this method. Ion-exchange chromatography reduced high background levels on gel scans used to quantitate the peptides; reduction with beta-mercaptoethanol substantially increased the efficiency of the cyanogen bromide cleavage; use of a concave gradient in acrylamide from 8 to 20% improved the resolution of cyanogen bromide peptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and a normalization procedure eliminated variations due to differences in the amount of material loaded on the gel system. This method of quantitation was applied to human aorta samples and to collagen secreted by human skin fibroblasts. Metachromasy of type I and type III collagen cyanogen bromide peptides stained with Coomassie blue R-250 was established and this was used as an index of the purity of the cyanogen bromide peptide preparations. Type I and III collagens were prepared from human placental tissue, and these purified collagens were used to construct calibration curves to determine the relationship between the quantity of diagnostic cyanogen bromide peptides present and the composition of the sample in terms of types I and III collagens.