An improved Coomassie Brilliant Blue (CBB R-250) staining to proteins in gels

An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported.The main improvement was using a fixing step of 25% trichloroacetic acid(TCA) before CBB staining.For most proteins studied,the sensitivity of the improved CBB staining was about twice as high as that of the typical method.For basic and low molecular weight proteins such as ribosomal proteins,the sensitivity of this improved staining method was about 3.5-28 times that of the typical method.It was speculated that the improved procedure would be suitable for exact quantitative analysis of proteins fractionated by SDS-PAGE,especially for basic and low molecular weight proteins.On the other hand,this new modified method might be also applied to multidisciplinary studies,such as biological researches and nuclear sciences.

Effects of Temperature on Determination of Protein Concentration with Coomassie Brilliant Blue Method

[Objective] This study aimed to investigate the effects of temperature on determination of protein concentration with Coomassie Brilliant Blue method,thus proving advice and guidance for accurate determination of protein concentration.[Method] With Coomassie Brilliant Blue method,the concentrations of different bovine serum albumin samples were determined under different temperatures and incubation time.[Result] According to the standard curve,when the determination range of protein concentration was 0-100 mg/ml,the determined protein concentration was relatively stable after incubation at 20 ℃ for 20-30 min.Furthermore,the determination result of higher protein concentration with Coomassie Brilliant Blue method was less affected by various factors.[Conclusion] In determination of protein concentration with Coomassie Brilliant Blue method,temperature,sample concentration and incubation time were important factors affecting the accuracy of experimental results.

2DGE-coomassie brilliant blue staining used to differentiate pasteurized milk from reconstituted milk

Differentiating pasteurized milk and reconsti-tuted milk by scientific approach was necessary to defend consumer from economic fraud of wrong labeling. In this paper 2DGE (2 Dimen-sional Gel Electrophoresis)-coomassie brilliant blue staining method was employed and sig-nificant color intensity changing was observed among raw milk, pasteurized milk, UHT milk and reconstituted milk. For example, the intensity of 10 protein spots including casein and lac-toglobulin reduced more than two folds from pasteurized milk to reconstituted milk. However, DIGE (Differential Gel Electrophoresis) assay showed that the majority protein remained simi-lar level from pasteurized milk to reconstituted milk. Therefore the color fading of coomassie brilliant blue stained 2D gels may be due to other biochemical reaction, such as Maillard reaction, instead of protein degradation. Stability of 2DGE pattern was confirmed by running six gels of the same sample in parallel and software analysis showed that all proteins were at similar level. Two commercialized pasteurized milk samples and one reconstituted milk sample were tested by 2DGE-coomassie blue staining method and re-constituted milk could be easily identified.

Non-decoloured In-gel Digestion of Coomassie Blue-stained Proteins

A simplified method is presented for tryptic digestion of Coomassie brilliant blue(CBB)-stained proteins in polyacrylamide gels. Compared with conventional methods, the proposed method does not require a removal of the dye before digestion, and is thus faster and saves a lot of labor. The resulted digest can be analyzed by either RPLC/ESIMS or MALDI MS for identification of the protein in a conventional way. Model studies with bovine serum albumin(BSA) showed that 50 ng of the protein could be routinely identified. The simplified procedure displays a tendency to produce more incompletely cleaved peptides, which is favorable for improving the sequence coverage.

尿蛋白定量方法探讨及对CBB法的改良

探讨了尿蛋白测定方法,并改良了CBB—I尿蛋白定量法,于试剂中加入SDS和TritcX-100,可改善不同比例白、球蛋白反应灵敏的差异。并观察了蛋白成份及温度对5种蛋白测定方法反应的影响。实验证明:CBB—Ⅱ法在许多方面优于其他方法,操作简单、快速,适于临床常规尿蛋白定量测定。

低电荷柱撑蒙脱土对考马斯亮蓝的吸附性能研究

采用微波法制得低电荷锂化蒙脱土(the reduced-charge lithiated bentonite,RCBT),再制备羟基铝柱撑剂,插层于RCBT中制备低电荷铝柱撑蒙脱土(the aluminum-pillared bentonite,Al/RCBT)。对Al/RCBT进行结构和形貌表征并考察它对考马斯亮蓝的吸附性能。研究表明:羟基铝柱撑剂成功插入RCBT层间,使蒙脱土的层间距增大了0.78 nm;最佳吸附pH值为2,吸附30 mg/L考马斯亮蓝溶液40 min即达到平衡,吸附容量达17 mg/g;Al/RCBT对考马斯亮蓝的吸附符合准二级动力学模型和Freundlich等温模型。

Biodegradation and detoxification of the triphenylmethane dye coomassie brilliant blue by the extracellular enzymes from mycelia of Lactarius deliciosus

Fungi play an important role in dying wastewater treatment.In this work,the mycelia of Lactarius deliciosus exhibited an excellent capacity in decolorizing coomassie brilliant blue(CBB).The results demonstrated that the mycelia could treat CBB with high concentrations over a broad range of pH and temperature.The decolorization rate of 99.19%and the removal rate of 16.31 mg·L^(‒1)·h were realized.The mycelia could be recycled from decolorizing process for 19 times,indicating a good re-usability.It verified that the lignin peroxidase(121.65 U·L^(‒1))and manganese peroxidase(36.77 U·L^(‒1))were involved in the degradation and decolorization process of CBB.Toxicity assessments indicated the seed germination rate was up to 82.22%while inhibition to Escherichia coli decreased dramatically and no significant effect on Caenorhabditis elegans growth was found.The removal of CBB was a synergistic process accomplished by adsorption and biodegradation.The mycelia could be used for eco-friendly CBB treatment.

考马斯亮蓝法测定核桃水溶性蛋白含量的研究

采用考马斯亮蓝G-250染色法对核桃粗蛋白中可溶性蛋白质含量进行了测定,结果表明,蛋白质浓度为10mg/mL^80mg/mL时与吸光度值有良好的线性关系,考马斯亮蓝G-250染色法简单、快速、灵敏,且相对较为准确,是测定低浓度蛋白质含量的有效方法。通过此方法,测得的核桃水溶性蛋白平均含量为8.37%。

考马斯亮蓝法测定果汁中蛋白质的含量

采用考马斯亮蓝法,对不同果汁中的蛋白质含量进行了测定。结果表明:蛋白质浓度在10-80μg/mL内与吸光度有良好的线性关系(R2=0.9964);样品平均回收率(n=3)分别为99.2%、98.8%、101.8%,RSD分别为0.64%、0.81%和0.58%(n=3)。该方法具有快速、灵敏度高、重现性好等特点。

考马斯亮蓝法测定苹果组织微量可溶性蛋白含量的条件优化

以红星苹果果肉为试材,探讨不同提取缓冲液(磷酸缓冲液、Tris-HCl缓冲液和水)、缓冲液pH值、浓度、外源添加物和料液比等因素对苹果果肉可溶性蛋白提取效率的影响。结果表明:外源添加PVP和EDTA以及料液比是苹果可溶性蛋白提取率的主要影响因素。实验最终确定的提取条件为0.1mol/L pH9.0 Tris-HCl提取缓冲液(内含1mmol/L EDTA和1%PVP)、料液比1:25,此条件下苹果果实可溶性蛋白的有效测定含量为0.544%,为水提组(0.073%)的7.45倍。

考马斯亮蓝染色法检测人精子顶体反应的评价

为建立一种检测人精子顶体反应的简便实用的新技术，用３．５％高氯酸水溶液配制０．０５％考马斯亮蓝（Ｒ２５０）染色液浸染人精子３０ｍｉｎ，顶体完整者顶体区染成紫蓝色，顶体反应者则不染。对获能前后和钙离子载体Ａ２３１８７诱导顶体反应的精子进行染色并与经典的顶体反应检测技术ＰＳＡ法对照，两种方法检测顶体反应率无显著差异。方法学检验证明新技术结果可靠。本法操作简便、经济，普通显微镜即可检测，易于推广，克服了原有技术复杂昂贵的缺点，具有研究和临床诊断的良好价值。

考马斯亮蓝法测定野木瓜多糖中蛋白质的含量

采用考马斯亮蓝染色法对野木瓜多糖中蛋白质含量进行测定,在波长595 nm处测定吸光度,实验表明标准蛋白质在1.67μg/mL￣16.67μg/mL之间呈现良好的线性关系,该法简便迅速,灵敏度高、重现性好,是测定野木瓜多糖中蛋白质含量的有效方法,并通过此法测得野木瓜多糖中蛋白质平均含量为12.17%。

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G2 5 0 (CBBG2 5 0 )染色方法 .该方法所用试剂仅为稀盐酸和CBBG2 5 0 ,CBBG2 5 0的工作浓度为 0 0 0 15 % ,灵敏度达 0 0 2 μg/带 ,染色 2h达 70 % ,4h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 。

考马斯亮蓝法快速测定乳品中蛋白质含量

采用考马斯亮蓝G-250染料染色法对市售乳品中蛋白质进行测定,在595 nm波长处测定溶液的吸光度。实验结果表明,标准蛋白质在10～80μg/mL之间呈现良好的线性关系。该法简便快速、灵敏度高、重现性好,是测定乳品中蛋白质的有效方法。

考马斯亮兰法(Bradford法)测定驼乳中蛋白质的含量

用考马斯亮蓝G-250显色,以牛血清蛋白为对照品,采用可见分光光度法在检测波长595 nm处测定驼乳中蛋白质的含量。结果表明,CBB G-250显色测定牛血清蛋白浓度在0.008~0.024 mg/m L（r=0.999 3）范围内与吸光度呈现良好的线性关系,加样回收率（100.20±2.25）%、RSD为2.24%。考马斯亮蓝显色法,具有简便、快速、灵敏度高、成本低、重现性好等特点,为新疆驼乳的进一步开发利用奠定基础。

几种双向凝胶电泳蛋白质检测方法的比较

本研究对蛋白质染色法进行比较和分析,以求获得满足蛋白质组研究所需的适宜的蛋白质检测法。实验采用2-D电泳分离急性早幼粒白血病细胞株NB4的全蛋白及1-D电泳分离蛋白质分子量标准物,从蛋白质检测的灵敏性、质谱兼容性、操作的简便性等角度,比较了传统考马斯亮蓝染色法、胶体考马斯亮蓝染色法、改良考马斯亮蓝染色法和银染法4种蛋白质着色法对凝胶分离蛋白质的检测影响,探讨了影响蛋白质染色与鉴定的影响因素。结果显示,银染检测灵敏度最高,但质谱鉴定相容性差,鉴定率近10%;改良考马斯亮蓝染色法检测灵敏度也高,可达4ng/spot,质谱鉴定率可接近55%。结论:改良考马斯亮蓝染色法的蛋白质检测灵敏度高,与质谱兼容好,能满足蛋白质组研究需要。

荧光光谱法和分子模拟技术研究考马斯亮蓝G-250与牛血清白蛋白的相互作用

采用多种光谱技术结合圆二色谱技术研究了考马斯亮蓝G-250(CBBG-250)相互作用于牛血清白蛋白(BSA),探究了两者之间的作用机理。荧光光谱结果表明BSA与CBBG-250结合形式是静态猝灭,随CBBG-250浓度增加,其形成常数随温度逐渐减小,结合比为1∶1,根据Stern-Volmer曲线计算焓变值(ΔH)和熵变值(ΔS)分别为-4.38kJ·mol^(-1)和-6.16J·mol^(-1)·K^(-1),均小于零,证明该过程是一个自发过程。傅里叶红外光谱测定结果表明CBBG-250的加入引起BSA结构的变化,随着CBBG-250溶液浓度的增加,BSA中色氨酸微环境极性被改变,在1 600~1 700cm^(-1)(酰胺带Ⅰ)和1 600~1 500cm^(-1)(酰胺带Ⅱ)处的特征吸收带蓝移。其中1 650cm^(-1)移动至1 710cm^(-1),1 544cm^(-1)移动到1 573cm^(-1),显示BSAα-螺旋(1 650~1 658cm^(-1))和β-折叠(1 620~1 640cm^(-1),1 675cm^(-1))结构发生变化,且α-螺旋含量比结合前有所降低。圆二色谱分析结果表明,两者间通过氢键和范德华力为主的作用力使得BSA二级结构发生变化,α-螺旋含量由42.15%下降至1.27%,该结果进一步被分子建模对接技术证明。

考马斯亮蓝G动力学光度法测定痕量铁的研究

研究了在稀盐酸介质中 ,α,α-联吡啶存在下 ,铁催化溴酸钾氧化考马斯亮蓝G,使其褪色这一新的指示反应及其动力学条件 ,建立了测定痕量铁的新方法。方法的检出限为 2 .2 6× 1 0 - 10 g/ m L,线性范围 0～ 0 .2 4μg/ 2 5 m L,用于食品及水中痕量铁的测定 。

考马斯亮蓝显色液组分对蛋白质测定的影响

考马斯亮蓝（ＣＢＢ）显色法测定蛋白质含量的主要缺点之一是线性关系差．通过研究显色液组分对线性关系的影响，发现显色液Ｈ＋浓度是影响线性关系的主要因素，并提出了一个新的显色液配方来改善考马斯亮蓝蛋白质测定法的线性关系．

测定亚硝酸根的新指示反应研究-氯酸钾-考马斯亮蓝G体系

基于稀盐酸介质中氯酸钾氧化考马斯亮蓝G的反应,提出了测定痕量亚硝酸根的新催化光度法.方法检出限为2.4×10～(-8)g/mL,线性范围为0.08～0.48μg/mL,用于唾液和硝酸钾中亚硝酸根测定.