蛋白免疫印迹法检测小分子蛋白的实验条件优化研究

目的:蛋白免疫印迹法自发明以来被广泛应用于现代生物学研究中的蛋白质定性和半定量分析。为了提高蛋白免疫印迹法的检测效率,需针对不同蛋白的特性调节相关的实验条件参数。本文旨在探讨免疫印迹法不同参数对小分子蛋白检测效果的影响,从而优化并获得最佳实验条件。方法:比较不同转膜电压和时间、转移缓冲液甲醇含量、不同化学发光剂对小分子蛋白的检测效果。结果:选择20 V、10 min转膜电压和时间所获得的信号显著高于10 V、25 min转膜条件,选择含20%甲醇转移缓冲液所获得的信号显著高于无甲醇转移缓冲液,选择飞克级化学发光剂所获得的信号显著高于纳克级化学发光剂。结论:选用高电压、短时间组合,选择含20%甲醇转移缓冲液和飞克级化学发光剂信号均有助于小分子蛋白免疫印迹检测。     

自制显示系统检测免疫印迹中的目的蛋白

目前蛋白免疫印迹显示系统有显色法和化学发光法,因化学发光法具有灵敏、快速、准确等特点而倍受科研工作者的青睐,但一般商品化的化学发光试剂盒价格颇为昂贵,为此,在参考文献的基础上作了一些改进,将Tris Cl的pH值从7.5提高到11.0,同时提高了对碘苯酚的浓度（从1mmol/L提高到2mmol/L）,得到了自制的化学发光试剂。首先用其检测了大肠杆菌中表达的2种不同分子量的蛋白（gam,13kDa;bet,30kDa）,发现当蛋白上样量为2~20ng时,各蛋白条带均清晰显示。随后的斑点免疫印迹实验表明：自制及进口化学发光试剂在检测酶标二抗效价方面有着同样高的敏感性。     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

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。     

黑豆多肽分离及其抗氧化活性的研究

采用凝胶柱层析法分离具有抗氧化活性的黑豆多肽,并分析其分子量分布;同时还采用DPPH·清除法研究各分离肽段的抗氧化活性,对抗氧化活性最佳的肽段进行动力学分析.结果表明:黑豆多肽经分离后得到3个片段,黑豆小分子肽BSP3对DPPH·自由基具有最强清除作用,其半清除浓度(HSC50)为0.664 mg/mL.所以,分子量小于1000 Da的黑豆小分子肽具有很好的抗氧化活性.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

兔骨组织总蛋白的提取和在免疫印迹法中的应用

探讨快速有效的骨组织总蛋白提取法和利用该方法进行免疫印迹研究.采用单纯研磨法、单纯锤击法和锤击研磨法分别对兔骨组织蛋白进行提取,通过聚丙烯酰胺凝胶电泳对蛋白进行分离,利用免疫印迹法检测骨组织中beta-actin的表达,比较3种骨组织蛋白提取法.发现3种蛋白提取法均可满足免疫印迹研究要求,但是锤击研磨法提取的蛋白含量比单纯锤击法高,并能更好的保留49 kDa以下的蛋白,而且该操作法比传统的单纯研磨法更为方便、快捷,还能节省液氛用量.锤击研磨法可作为提取骨组织蛋白的一种理想方法.     

骆驼蓬种子中一种具抗肿瘤活性蛋白的分离纯化及鉴定

骆驼蓬种子经浸提、硫酸铵沉淀、CM阳离子交换层析和Superdex 75凝胶过滤层析分离纯化得到一种具有抗肿瘤细胞增殖活性的蛋白(命名为PhLTP),经Tricine-SDS-PAGE检测为单一蛋白条带,高效液相色谱检测其表观分子量为14.8 kDa左右,表明PhLTP是由两条相同的亚基组成的蛋白.采用Edman降解法对该纯化蛋白的N-末端进行氨基酸测序,其N-末端序列与其他植物非特异性脂转移蛋白相似.对PhLTP抗肿瘤活性进行研究,结果表明其对HeLa、Eca-109、MGC-9和BEL-7404细胞都有增殖抑制活性,其中对HeLa细胞增殖的抑制作用较好,并具有浓度和时间依赖性,其IC50为45 μg/mL.通过Hoechst33258染色观察细胞形态,发现PhLTP能诱导HeLa细胞发生凋亡.     

榆黄蘑中一种抗病毒蛋白的纯化及其抗TMV和HBV的活性

采用阴离子交换层析和凝胶层析方法从新鲜食用菌榆黄蘑 (Plearotuscitrinopileatus)中进行了抗病毒蛋白的纯化 ,结果获得了一个纯化蛋白YP4 6 4 6 ,经SDS PAGE可确定其分子量为 2 7.4kD。以半叶法在枯斑寄主心叶烟上检测该蛋白对烟草花叶病毒 (TMV)的抑制率 ,发现有较好的抗TMV活性 ,其抑制TMV的中浓度为 0 .2 4 μg/mL。同时以HepG2 .2 .2 .15细胞株为模型 ,对所获得的蛋白进行体外抗乙型肝炎病毒效果的评价。结果表明该蛋白对HBsAg的 5 0 %抑制时的浓度为 0 .0 8μg/mL ,但对HBeAg效果不大     

杏鲍菇抗烟草花叶病毒蛋白的筛选

采用离子交换层析和凝胶层析方法,从杏鲍菇干样中分离得到多个蛋白组分,经枯斑寄主检测,发现多个蛋白组分都有抗烟草花叶病毒(TMV)的活性,对TMV的抑制率均在70%以上,高者可达99%。其中xb68Ab已得到了纯化,分子量约为23.7kD,在心叶烟和苋色藜上它对TMV侵染的抑制率分别达到99.43%和98.9%。     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3-10, 18 cm x 20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (> or = 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (< or = 40 kDa). Three hundred and sixty microg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm x 67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 microg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.     

不同浓度和梯度的SDS-PAGE胶对双向电泳中蛋白分离的影响

目的：探讨双向电泳中不同浓度和梯度的SDS-PAGE胶对肠道菌蛋白分离效果的影响。方法：制备弗氏2a志贺菌2457T野生株37℃晚期全菌蛋白质样品,进行不同浓度及梯度的SDS-PAGE,研究分离肠道菌蛋白最适宜的SDS-PAGE胶浓度。结果：获得了3个不同浓度（10%、12.5%和15%）的均一胶电泳图谱和3个不同梯度（4%～15%、10%～20%和12%～14%）的电泳图谱,并比较了这些图谱的分离效果;同时,为了分析肠道菌天然表达蛋白的相对分子质量范围,鉴定了8个极端相对分子质量蛋白。结论：对于肠道菌蛋白质的分离来说,12.5%的均一胶或12%～14%的梯度胶较为适宜。     

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.     

An automated in‐gel digestion/iTRAQ‐labeling workflow for robust quantification of gel‐separated proteins

Simple protein separation by 1DE is a widely used method to reduce sample complexity and to prepare proteins for mass spectrometric identification via in‐gel digestion. While several automated solutions are available for in‐gel digestion particularly of small cylindric gel plugs derived from 2D gels, the processing of larger 1D gel‐derived gel bands with liquid handling work stations is less well established in the field. Here, we introduce a digestion device tailored to this purpose and validate its performance in comparison to manual in‐gel digestion. For relative quantification purposes, we extend the in‐gel digestion procedure by iTRAQ labeling of the tryptic peptides and show that automation of the entire workflow results in robust quantification of proteins from samples of different complexity and dynamic range. We conclude that automation improves accuracy and reproducibility of our iTRAQ workflow as it minimizes the variability in both, digestion and labeling efficiency, the two major causes of irreproducible results in chemical labeling approaches.     

Erratum to “Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis”

Western blots are widely used for analysis of the expression levels of specific proteins. Blotting is conducted after sodium dodecyl sulfate or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels (one of which is stained) usually must be prepared, leading to the consumption of precious sample. Thus, we have developed a convenient and efficient Western blot method using a stained gel. This simple modification should be beneficial for the analysis of samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.     

Detection of myofibrillar proteins using a step gradient minigel with an ambiguous interface

Myosin heavy chain (MHC), actin, titin, and nebulin are four major myofibrillar proteins that interact with each other. However, it is difficult to analyze the four proteins simultaneously on the same minigel due to their broad range of molecular weights. Numerous gradient gels are normally used to detect these myofibrillar proteins. The conventional step gradient gel provides better separation of the four major proteins, but several proteins accumulate at the interfaces between different gradient layers. To eliminate the obvious interfaces, we employed a plastic syringe filled with 12 and 4% acrylamide solutions simultaneously and then established an improved step gradient minigel with an ambiguous interface. It was determined by blue dextran in-gel visualization and scanning densitometry that the acrylamide concentration at the ambiguous interface gradually changed. Coomassie blue staining and immunoblotting revealed that the four proteins were successfully separated and transferred for analysis. This gel system is simple to prepare and easy to use, and it is a reliable method for analyzing myofibrillar proteins or other protein mixtures with broad molecular masses.     

Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.     

Influence of gel dimensions on resolution and sample throughput on two-dimensional gels

To achieve high throughput and economical format of 2-D PAGE, comparison between gel size and resolution was conducted on human breast carcinoma cell line (MCF-7/AZ) proteins. SDS gel length showed a weaker influence of separation length on resolution in the second dimension, and there was little benefit of separation distances greater than 15 to 19 cm. IPG strip separation distances were very important with dramatic increase in resolution of longer gels compared with smaller gels, and maximal resolution was obtained using 18- and 24-cm IPG strips. Loading optimal amount of proteins on 2-D gels can also increase the number of detected spots. Therefore, taken together, compromise 2-D gels are crucial for higher capacity and higher throughput.     

Proteome analysis differentiates between two highly homologues germin-like proteins in Arabidopsis thaliana ecotypes Col-0 and Ws-2

A proteome approach based on 2-D gel electrophoresis (2-DE) was used to compare the protein patterns of the Arabidopsis ecotypes Col-0 and Ws-2. In leaf extracts a pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis after separation of protein extracts by 2-DE confirmed results from Coomassie blue-stained gels and revealed additional immunoreactive spots for both ecotypes most likely representing dimers of the spots first identified. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of AtGER3 protein in root extracts. Our results demonstrate the capacity of proteome analysis to analyse and distinguish closely related members of large protein families.