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。     

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.     

一种简易的与考马斯亮蓝相结合的低背景银染法

1972年Kerenyi建立的凝胶电泳的银染色法由于灵敏度高而被广泛采用。此法创建初期常产生较深的背景染色,以致影响弱带的显示,后来几经改进,特别是Blum等人利用硫代硫酸钠的化学特性,发展了一种高灵敏度的低背景银染方法,使此法愈     

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-...     

考马斯亮蓝法检测重组碱性成纤维细胞生长因子

目的：建立考马斯亮蓝法（CS）检测重组碱性成纤维细胞生长因子（r-bFGF)含量。方法：以牛血清白蛋白（BSA）为标准液,考马斯亮蓝（CBG－250）为染色剂,检测595nm吸光度并绘制标准曲线找出回归方程,对照r-bFGF样品计算含量。结果：此法敏感性高,可测出μg水平蛋白,标本量只需50－100μl,染料与蛋白的结合反应快,只需2min即可比色,显色稳定,可维持1h,重现性好,变异系数在5％以内。结论：此法简便有效、灵敏性高,能快速检测r-bFGF含量。     

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。     

蛋白质组学实验中聚丙烯酰胺凝胶的考马斯亮蓝染色

在蛋白质组学实验中,蛋白质经聚丙烯酰胺凝胶电泳后的染色是一个十分重要的环节。鉴于其良好的质谱兼容性和操作上的便捷,考马斯亮蓝染色是目前众多实验室中最常用的染色方法。本文介绍了四种各具特色的考马斯亮蓝染色方法。     

粗毛栓菌产漆酶对考马斯亮蓝的脱色降解

粗毛栓菌粗酶液经聚丙烯酰胺凝胶电泳,在CBBG－250染色后,漆酶带处有一透明圈,经纯化漆酶和CBBG－250溶液直接作用以及菌体的培养结果证实,漆酶对CBBG－250具有一定的脱色降解作用,在漆酶活力达118u/ml时,CBBG－250溶液在595nm波长的光吸收60min内下降了32．4％。该粗毛栓菌产漆酶对工业染料废水也具有一定的降解脱色作用。     

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。     

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.     

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.     

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.     

Reproductive and Neurobehavioral Effects of Brilliant Blue FCF in Mice

Brilliant blue FCF of food color was given in the diets of mice at levels of 0% (control), 0.08, 0.24, and 0.72% from 5 weeks of age in the F₀ generation and continuing to 11 weeks of age in the F₁ generation and selected reproductive and neurobehavioral parameters were measured. Mice were mated at 9 weeks of age and dams were delivered offspring at 12 weeks of age. Offspring were weaned at 4 weeks of age. Regarding exploratory behavior at 8 weeks of age in the F₀ generation, movement time (sec) displayed a significant tendency to be increased and the average time of rearing (sec) displayed a significant tendency to be decreased in females in the treatment groups in a trend test (p = 0.019 and 0.027, respectively). In the F₁ generation, the development of surface righting at postnatal day 4 was delayed significantly in the high‐dose group (0.72%) in male and female offspring, and those effects were significantly related to dose in a trend test (p< 0.01 for both). Regarding exploratory behavior at 8 weeks of age in the F₁ generation, the number of horizontal activities exhibited a significant tendency to be decreased in females in the treatment groups in a trend test (p = 0.015). Regarding spontaneous behavior, average time of movement (sec) was significantly accelerated in females in the high‐dose group. The dose levels of brilliant blue FCF used in the present study produced a few significant effects on neurobehavioral parameters in multiple generations in mice.     

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。