一种简便的考马斯亮蓝G250蛋白质染色方法

介绍一种快速、简便、几乎无背景的考马斯亮蓝G250(CBB G250)染色方法.该方法所用试剂仅为稀盐酸和CBB G250, CBB G250的工作浓度为0.0015%,灵敏度达0.02 μg/带, 染色2 h达70%,4 h以上或染色过夜即可充分染色.与以往的考马斯亮蓝染色方法相比,该方法有经济方便、灵敏度高、几乎无背景等优点,便于推广应用.     

聚丙烯酰胺凝胶电泳的快速脱色方法

以牛血清白蛋白为材料进行聚丙烯酰胺凝胶电泳(PAGE)和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),凝胶固定后,用考马斯亮蓝R-250染色后比较传统脱色液(冰乙酸-甲醇溶液)和不同盐溶液(NACL、KCL、CUCL2)的脱色效果的结果表明:PAGE和SDS-PAGE胶,0.25和0.5MOL·L-1NACL,在70℃(PAGE)、50℃(SDS-PAGE)下脱色,约2￣4H,效果好,灵敏度高,背景低。     

改良的蛋白质电泳考马斯亮蓝染色方法研究

旨在建立简便、经济、灵敏以及质谱兼容的蛋白质聚丙烯酰胺凝胶电泳染色法,在胶体考染(Blue Silver)配方(0.12%的考马斯亮蓝G-250,10%的磷酸,10%的硫酸铵,和20%的甲醇)的基础上,通过优化凝胶固定时间与染色液中各物质用量,得到改良的染色液配方(0.1%的考马斯亮蓝G-250或R-250,5%的磷酸,5%的硫酸铵,10%的甲醇)及简便的操作步骤。试验对比结果显示,改良方法一与胶体考染相比仍保持了较高的染色灵敏度以及较好的质谱兼容性,且有机试剂使用量更少,染色深度与蛋白质量的线性关系也更好。     

蛋白质定量方法的进展

本文在简要比较常用蛋白质定量方法的基础上,结合自己的工作,选择性地叙述了Lowry法、考马斯亮蓝G-250染料测定蛋白质的改进法。还介绍了银染色定量法和4-甲酸喹啉测定蛋白质含量的新方法。     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。     

考马斯亮蓝G-250染色法测定柠条锦鸡儿种子中可溶性蛋白含量

目的:建立快速、灵敏的蛋白质含量测定方法,对柠条锦鸡儿种子中可溶性蛋白质含量进行测定;方法:运用超声与研磨两种方法提取柠条锦鸡儿种子中的可溶性蛋白,采用考马斯亮蓝G-250染色法对其进行测定;结果:超声提取与研磨提取得到可溶性蛋白含量分别为6.91%、6.87%,RSD分别为0.72%,0.67%;结论:考马斯亮蓝G-...     

一种简单快速的聚丙烯酰胺凝胶电泳染色法——铜染色法

十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)作为分离鉴定生物大分子的有效手段已广泛用于分子生物学和医学临床工作,迄今为止,用于该技术的染色法多沿用考马斯亮蓝(CBB)和银染色。但上述两法均有其局限性,如蛋白凝胶染色前须经酸醛固定,不易洗脱且操作繁琐,脱色耗时。最近,Lee等报道一种SDS-PAGE负染色法,利用氯化铜分别与凝胶中     

聚丙烯酰胺凝胶中蛋白质快速染色的改良法

聚丙烯酰胺凝胶中蛋白质的染色常采用考马斯亮蓝R250为染料进行染色,此法不仅操作繁琐、费时,而且由于需要脱色,很容易将一些染色较弱的蛋白区带颜色褪去,使之不能观察,灵敏度只能达到0.25μg。Reisner等人报道了以考马斯亮蓝G250为染料的快速简便染色方法,它能迅速地观察到凝胶中     

考马斯亮蓝—银染色法——一种更灵敏的凝胶蛋白质染色法

我们曾报道一种重复性好而灵敏的电泳凝胶蛋白质染色法。其后发现银染色法虽然灵敏,但有一定的选择性,某些蛋白不易被银染色着色。若将考马斯亮蓝染色与银染色相结合,则可弥补彼此弱点,而且可提高灵敏度,是一种较为理想的凝胶蛋白质染色法。一、材料与方法(一)试剂丙烯酰胺、双丙烯酰胺、SDS 为国产品,均重结晶后使用;考马斯亮蓝 G—250及戊二醛(Fluka 产品);硝酸银 GR 级(西安化学试剂厂产品);Ampholine(LKB 产品);尿素     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.Several improvements of the original Coomassie protocol¹ have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution², and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol³. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff''s colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol⁴. The novel aluminum-based staining in Kang''s study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.Here, we demonstrate application of Kang''s protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.Download video file.^{(128M, mp4)}     

CGP stain: An inexpensive, odorless, rapid, sensitive, and in principle in vitro methylation-free Coomassie Brilliant Blue stain

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12 ng within 45 min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation.     

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.     

Laccase-mediator system in the decolorization of different types of recalcitrant dyes

Phloroglucinol, thymol, and violuric acid (VIO) were selected as laccase mediators after screening 14 different compounds with indigo carmine (indigoid dye) as a substrate. With the presence of these three mediators, a nearly complete decolorization (90-100%) was attained in 1 h. Thus, these three compounds were used as mediators for the decolorization of other four dyes. The results indicated that VIO was effective mediator in decolorization of Remazol brilliant blue R (RBBR, anthraquinoid dye) and Coomassie brilliant blue G-250 (CBB, triphenylmethane dyes), and Acid red (diazo dye). In presence of VIO, the four dyes described above attained 70% decolorization. Thymol was able to mediate decolorization of RBBR and Azure A (heterocyclic dye). Phloroglucinol has no mediating capability in decolorization of the four dyes analyzed. Mediator concentration, pH, and copper ion have an effect on the decolorization of the RBBR. Our data suggested that the decolorization capabilities of laccase/mediator system were related to the types of mediator, the dye structure and decolorization condition.     

稻曲病菌厚垣孢子不同破壁方法的比较

为了探究稻曲病菌[Ustiloginoidea virens(Cooke)Takahashi]厚垣孢子的最佳破壁方法,研究采用4种破壁法对该病菌黄色和黑色厚垣孢子进行破壁,血球计数板计算破壁效果,并用考马斯亮蓝法测定不同破壁方法中厚垣孢子壁内可溶性蛋白含量。结果表明,在普通光学显微镜下观察,破壁后厚垣孢子多数为碎片,少数为孢壁内空圆球。4种破壁方法中液氮研磨-超声破碎法破壁效果最好,黄色和黑色厚垣孢子的破壁率均可达98%以上,用该法破壁测得的黄色和黑色厚垣孢子壁内可溶性蛋白质含量也最高。由此可见,液氮研磨-超声波破碎法是一种稻曲病菌厚垣孢子破壁的有效、简便、适宜在实验室应用的方法。     

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.     

Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Unusual gangliosides of eggs and embryos of the sea urchin Strongylocentrotus intermedius. Structure and density-dependence of surface localization

From eggs and embryos of the sea urchin Strongylocentrotus intermedius two gangliosides, provisionally named G-1 and G-2, were isolated in the pure state. Both gangliosides contained glucose, N-glycoloylneuraminic acid and sphingosines in a 2:2:1 ratio; G-2 contained also a sulfate group, and yielded G-1 on desulfation. By periodate oxidation/borohydride reduction, permethylation analysis, neuraminidase degradation, analysis of the aldohexitol acetates and mass-spectrometry G-1 and G-2 were shown to have hitherto unknown structures: G-1 was identified as N-glycoloylneuraminosyl-(alpha 2 leads to 6)-glucosyl-(1 leads to 8)-N-glycoloylneuraminosyl-(2 leads to 6)-glucosyl-(1 leads to 1)-ceramide, and G-2 as sulfated G-1, carrying a sulfate ester group at C-8 of the terminal sialic acid. Antisera against the two gangliosides were prepared in rabbits by immunization with ganglioside G-1 or G-2. The specificity of the antisera was revealed by immunoelectrophoresis and immunodiffusion. The antisera did not react with bovine-brain and rat-liver gangliosides, with glucosylceramide and with various hydrolytic fragments of G-1 and G-2. The surface localization of the gangliosides in embryos incubated at different cell densities was studied by immunofluorescence microscopy. The intensity of the immunofluorescence was found to increase with decreasing cell density, indicating a different surface organization in sparse and dense embryos. In the sparse embryos immunofluorescence was seen mainly in the contact regions between the blastomers.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)