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。     

桔小实蝇幼虫总蛋白双向电泳技术体系的建立和优化

【目的】建立和优化桔小实蝇幼虫Bactrocera dorsalis(Hendel)总蛋白的双向电泳条件。【方法】使用BPP法和3种TCA-丙酮法(TCA-丙酮-A法:直接加入裂解液磨样;TCA-丙酮-B法:样品提取液中加入40 mmol/L DTT;TCA-丙酮-C法:样品提取液中加入0.07%β-巯基乙醇)提取桔小实蝇幼虫总蛋白;使用13 cm和24 cm p H 4~7的IPG胶条分离桔小实蝇幼虫总蛋白;使用考马斯亮蓝法及硝酸银染色法对双向电泳凝胶进行染色;使用5800 MALDI-TOF-TOF MS/MS质谱分析仪对BPP法获得的特异蛋白进行质谱鉴定,并将检索数据库物种分别设为Metazoa(Animals)与Drosophila(Fruit flies)进行数据库检索。【结果】TCA-丙酮法中,TCA-丙酮-C法提取效果最好,BPP法优于所有TCA-丙酮法;使用考马斯亮蓝染色与硝酸银染色效果相当;使用24 cm胶条的蛋白分辨率明显高于13 cm胶条;检索数据库物种设为Metazoa(Animals)可获得比Drosophila(Fruit flies)更加全面的信息。【结论】使用24 cm p H 4~7的IPG胶条对BPP法提取的桔小实蝇幼虫总蛋白进行双向电泳,采用考马斯亮蓝法对双向电泳凝胶进行染色,可得到更好的双向电泳图谱,检索数据库时检索物种可优先设为Metazoa(Animals)。     

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     

一种利用普通垂直电泳槽回收PAGE胶蛋白条带的简便方法

植物总蛋白样品经聚丙烯酰胺凝胶电泳分离之后,直接用考马斯亮蓝染色、切胶回收目的条带,再用聚丙烯酰胺凝胶电泳槽电洗脱纯化得到单一条带的目的蛋白.此法可得到有活性的黄瓜衰老叶片中被特异激活的DNA酶,对样品中含量少,特别是与其他分子量相近的蛋白质十分有效.     

一种利于蛋白质回收的快速SDS-聚丙烯酰胺凝胶电泳染色-脱色方法

在实践基础上摸索出一种快速并有利于蛋白质回收的SDS-聚丙烯酰胺凝胶电泳染色-脱色方法：经常用的考马斯亮蓝R-250染色液短暂染色后直接用250 mmol/L KCl溶液脱色.这种方法染色-脱色的清晰程度与常规的染色-脱色方法接近,而且能使蛋白质回收率提高三倍多.     

一种转移非特异性蛋白带的染色方法

考马斯亮蓝是常用的聚丙烯酰胺凝胶蛋白电泳的染料,利用硝酸纤维素膜(NCF)对染料的吸附作用,将低浓度的考马斯亮蓝(0.025%)染色液直接对NCF上的转移蛋白带进行染色,经实验反复验证.它是一种较好的NCF上转移非特异性蛋白带的染色方法.     

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。     

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.     

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-...     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。     

茶树叶糖蛋白分离及鉴定的初步研究

茶叶是一种有益健康的保健食品,保健功能在于它有多种化学活性成分。近年来研究表明,茶叶糖蛋白可能是茶叶的保健因子之一。从茶树叶中提取了总蛋白、45%~70%硫酸铵分级总蛋白、分级蛋白Sephadex G-100凝胶层析分离出粗糖蛋白,根据糖蛋白糖链及蛋白的对照染色鉴定出多种茶树叶糖蛋白,并从SDS-胶上快速纯化了三种茶叶糖蛋白。ConA-Sepharose 4B亲和层析从茶树叶总蛋白中分离出16种天然活性糖蛋白。     

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.     

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.     

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.     

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.     

Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-d gels. A comparison of staining with coomassie brilliant blue and sypro ruby

When separating protein mixtures on 2-D gels for proteomics purposes, fluorescent staining is superior in sensitivity and linear response as compared to Coomassie Brilliant Blue (CBB) and silver staining, respectively. We have compared the quality of mass spectra for proteins obtained from gels stained with CBB and SYPRO Ruby (SR) and found significant differences. These differences can be seen both in inferior signal/noise ratios and number of peptides detected with the fluorescent stain.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

蝴蝶兰叶片蛋白质提取及双向电泳体系优化

通过对蛋白质提取、IPG胶条选择、上样量、水化方式、聚焦条件等方面的优化,建立蝴蝶兰叶片蛋白质的双向电泳体系。结果表明,采用酚抽提法提取蝴蝶兰叶片蛋白质的纯度较高,复溶较完全;双向电泳优化体系选用24 cm pH 3～10 NL的IPG胶条,被动水化,上样量为1.35 mg,B1程序进行等电聚焦,12%分离胶进行第二向电泳,考马斯亮蓝G-250染色。该方法获得分辨率较高、重复性较好的蝴蝶兰叶片双向电泳图谱,蛋白数点多达1163个,可以满足蝴蝶兰蛋白质组学研究和分析。     

Evaluation of protocols used in 2-d electrophoresis for proteome analysis of young rice caryopsis

In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) of proteins, including gel staining protocol, pH range of immobilized pH gradient (IPG) strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate (reported by Heukeshoven and Dernick in 1988) and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid (reported by Pink et al in 2010) demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130 μg using the 17 cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis.     

蛋白负染方法在蛋白质组学中的应用评价

为评价蛋白质负染方法在蛋白质组学分析中的应用,采用负染和考马斯亮蓝染色两种方法对同一样品的双向电泳胶进行染色,取相对应的8对蛋白点,并进行胶内酶解及MALDI-TOF/TOF分析,比较两种方法与质谱的兼容性。图像分析显示,负染方法展示出的蛋白点更多,但三维峰图不如考染明晰;质谱结果显示,8个负染蛋白点中有7个鉴定结果有效,8个考染蛋白点鉴定结果均有效。因此可以得出以下结论:负染的灵敏度高于考染,与质谱的兼容性良好,适用于建立双向电泳参考图谱的研究;但负染后的胶图不适于进行蛋白点丰度对比分析。