西花蓟马蛋白的提取及双向电泳体系的建立

【目的】蛋白样品的制备是获得良好双向凝胶电泳(2-DE)图谱的前提,建立合理的西花蓟马蛋白的双向电泳体系,获得分辨率较高、重复性较好的图谱,能够为后续的研究提供有力支撑。【方法】实验以西花蓟马成虫为实验材料,对比了饱和酚法、TCA/丙酮法和直接裂解法3种蛋白提取方法,从中选出最适宜双向电泳分析的一种蛋白提取方法。【结果】3种方法蛋白提取率差异显著,直接裂解法蛋白提取率最高,饱和酚法的蛋白提取率最低;3种方法的SDS-PAGE条带数差异不明显;TCA/丙酮法的双向凝胶图谱效果最好,蛋白点最多。【结论】TCA/丙酮法能够有效去除西花蓟马蛋白中的干扰物质,是最适合西花蓟马双向凝胶电泳的蛋白提取方法,为后续西花蓟马在蛋白组学方面的研究奠定了基础。     

不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPGpH3～10,24cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。     

灵芝子实体原基双向电泳和总蛋白质提取方法的建立

比较了Tris-饱和酚法和三氯乙酸(TCA)/丙酮沉淀法对灵芝子实体原基总蛋白质的提取效果。用Image Master 2D Platinum6.0软件分析两种方法所提蛋白质的双向电泳图谱,分别得到565和273个蛋白质点;(TCA)/丙酮沉淀法在碱性端低分子量区域有些蛋白质斑点存在拖尾现象,Tris-饱和酚法能有效去除样品中的盐分,使蛋白质的聚焦效果更好,蛋白点数增加。Tris-饱和酚法可做为灵芝子实体原基总蛋白质的提取方法并为其他药用真菌总蛋白质的提取提供参考,同时为本实验室后续研究灵芝双向性固体发酵雷公藤的蛋白差异表达奠定了基础。     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

适用于盐生植物的双向电泳样品制备方法

比较了三氯乙酸,丙酮沉淀法（TCA）、三氯乙酸沉淀法（E-TCA）和酚抽法（Phe）3种方法对盐生植物盐角草（Salicornia europaea L．）总蛋白的提取效果。3种方法分别得到579、343和535个蛋白点;TCA和E-TCA法所得图谱均存在严重的横向纹理,Phe法所得图谱则背景干净,基本上没有纹理。说明Phe法不仅能很好地提取盐角草蛋白,而且能有效去除样品中的盐分。对Phe法的提取液进行了改进,所得图谱背景更加清晰,蛋白点数增加。为其他盐生植物以及嗜盐微生物蛋白质的提取提供了重要参考。     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.     

Proteomic study of a model causative agent of harmful red tide,Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis

A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.     

巴两橡胶树胶乳黄色体蛋白提取及双向电泳方法初探

巴西橡胶树(Hevea brasiliensis)的黄色体在胶乳凝固和保护植株过程中有重要作用。本文比较使用三氯醋酸/丙酮(TCA/ ACE)、Tris缓冲液、磷酸缓冲液提取橡胶树胶乳黄色体总蛋白的双向电泳效果。确定一种适合双向电泳的蛋白提取方法。结果表明Tris缓冲液提取法得到的双向电泳图谱可以达到300个,尤其是低丰度蛋白呈现性较好,适合提取黄色体蛋白以进行双向电泳。     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

巴西橡胶树胶乳黄色体蛋白提取及双向电泳方法初探

巴西橡胶树（Hevea brasiliensis）的黄色体在胶乳凝固和保护植株过程中有重要作用。本文比较使用三氯醋酸/丙酮（TCA/ ACE）、Tris缓冲液、磷酸缓冲液提取橡胶树胶乳黄色体总蛋白的双向电泳效果。确定一种适合双向电泳的蛋白提取方法。结果表明Tris缓冲液提取法得到的双向电泳图谱可以达到300个,尤其是低丰度蛋白呈现性较好,适合提取黄色体蛋白以进行双向电泳。     

A high-efficiency,two-dimensional gel electrophoresis platform for mature leaves of grass pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Grass pea (Lathyrus sativus L.) is the most drought-tolerant legume crop rich in dietary protein. However, little is known about the molecular mechanisms of its drought resistance. Two-dimensional gel electrophoresis (2-DE) is an important experiment technique in proteomics, which has been applied extensively in studies on plant resistance to abiotic stress. To establish an effective 2-DE platform and further study the drought-resistance mechanisms of grass pea using proteomic approaches, three protein extraction methods, different isoelectric focusing (IEF) conditions and various types of gel strips were evaluated using mature leaves. The results showed that the trichloroacetic acid (TCA)/acetone protein extraction method, extending time at low voltage for IEF and using 18 cm gel strip with pH 4.0–7.0 were optimum conditions for 2-DE analysis of grass pea leaves. Applying these optimized 2-DE conditions, 1,481 total protein spots were detected in control leaves and 1,346 spots in polyethylene glycol -treated leaves, of which 67 differentially expressed protein spots were obtained relative to the control. These data suggested that an efficient 2-DE platform with high repeatability and resolution for grass pea mature leaves had been established for the first time here, which could be further used to investigate the drought-resistance molecular mechanisms of grass pea.     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

荔枝果皮总蛋白质提取及双向电泳体系的建立

用TCA-丙酮、丙酮和酚3种方法提取荔枝(Litchi chinensis Sonn.)果皮的总蛋白质,比较了蛋白产量、单向SDS-PAGE和双向电泳等方面的差异,并对双向电泳体系进行探索.结果表明:酚抽提法最佳,提取的总蛋白得率最高,蛋白在单向SDS-PAGE中形成条带数目最多,最清晰;经双向电泳分离用银染显色,可检...     

柑橘叶片和果皮总蛋白质提取及双向电泳体系的建立

以春甜橘(Citrus reticulata Blanco‘Chuntianju’)果皮和叶片为材料,分别用Tris-HCl、尿素/硫脲(Thi/Urea)、三氯乙酸/丙酮(TCA)和酚(Phe)等4种方法提取柑橘总蛋白质,从蛋白质产量、单向SDS-PAGE和双向电泳等方面进行比较。结果表明,4种方法的分离效果存在较大差异,不论是以柑橘叶片还是果皮为材料,均以TCA法最好,且双向电泳图谱分辨率较好,蛋白点清晰、均匀、基本没有条纹,且蛋白点多。这说明TCA法不仅能很好地去除柑橘果皮、叶片中存在的大量干扰物质,而且还能得到稳定的蛋白点。     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi

The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction?was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium?perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.     

板栗疫病菌致病性机理的双向凝胶电泳法研究

双向凝胶电泳技术是蛋白质组学研究的基础性技术平台。如何得到一张高质量的双向凝胶电泳图谱是进行后续研究的关键。为探索适用于板栗疫病菌可溶性总蛋白的最佳提取条件,从蛋白组学角度来探索板栗疫病菌致病性机理,比较了目前在丝状真菌中常用的两种蛋白质提取方法,制备的蛋白质样品经双向凝胶电泳后,在凝胶上呈现的蛋白质斑点的丰度和分布特点。结果表明,两种方法获得的蛋白质主要集中分布在pH4~7的范围内;TCA-丙酮沉淀法得到的图谱分辨率高但是蛋白质总量很少。裂解液-TCA-丙酮沉淀法得到的蛋白质总量较大,通过cleanupkit处理后图谱分辨率可以达到差异蛋白组的要求。随机提取几个银染蛋白点用MALDI-TOFMS/MS进行分析,可以得到高质量的肽质量指纹谱。表明该样品制备方法可以满足蛋白质鉴定的要求。     

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.