小麦苗期地下茎蛋白质双向电泳技术体系的优化

以东农冬麦1号为材料,对苗期地下茎处的蛋白提取方法、蛋白溶解、上样量、胶条的转移等方面进行了试验.结果表明:在蛋白提取方面,TCA/丙酮法(T法)和尿素/硫脲法(N法)相比T法能减少低丰度蛋白的损失得到蛋白点数更多的图谱.在蛋白溶解方面,经过两次水化液溶解的蛋纯度较高,在等电聚焦时能保持8000伏较高电压.上样量方面,10mg粗蛋白溶于两次水化液能得到清晰、分离效果好、蛋白点数较多的图像.胶条转移方面,先向胶面中加入400μl0.3%普通琼脂糖溶液后,用200μl的电极缓冲液冲洗胶条支撑膜会使胶条顺利转移到第二向胶面上且胶条与胶面间不会产生气泡.     

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。     

适用于黄麻根部蛋白质组学分析的双向电泳技术

以黄麻品种'9511'幼苗为试验材料,研究其根部蛋白提取方法的得率及不同的蛋白样品溶解方法、电泳上样量和IPG胶条pH范围对双向电泳图谱的影响.结果表明:采用三氯乙酸(TCA)/丙酮沉淀法提取黄麻根部蛋白质,蛋白得率为80 mg/g;蛋白粉末溶解采用两次水化法,裂解液中含有7 mol/L尿素、2 mol/L硫脲、4% CHAPS、65 mmol/L DTT、0.2%载体两性电解质和1 mmol/L PMSF,能够较充分地溶解蛋白质,且制备的样品浓度能够满足双向电泳上样要求;上样量为400 μg时得到的图谱分辨率高、蛋白斑点分布均匀、清晰;等电聚焦(Isoelectrofocusing,IEF)采用pH 4～7、17 cm的IPG胶条时所得图谱质量最佳.研究表明,样品的制备及IEF有效除盐对获得理想的2-DE图谱非常关键;取材、染色等细节对2-DE的重复性影响很大.     

汾酒大曲宏蛋白质组的制备与双向电泳技术的建立

采用TCA-丙酮沉淀法、丙酮沉淀、硫酸铵等沉淀法制备汾酒大曲的宏蛋白组样品,并用双向电泳来检测制备效果,结果表明：TCA-丙酮沉淀法制备的样品经电泳分离后,减轻了杂质干扰,其2-DE图谱中竖条纹干扰较少,且获得的蛋白点形状规则、清晰且无明显重叠现象,优于其它两种方法;经过两次水化液溶解的蛋白样品在等电聚焦时能保持4000 V较高电压;上样量为300μg左右的粗蛋白溶于二次水化液能得到点数更多、分辨率高的电泳图谱。建立了汾酒大曲宏蛋白质组的双向电泳体系,为汾酒品质研究奠定基础。     

四倍体刺槐枝条韧皮部总蛋白质双向电泳体系建立及应用

采用TCA/丙酮法对四倍体刺槐枝段韧皮部全蛋白质进行提取,通过对IPG胶条pH梯度、分离胶浓度的选择,上样量、等电聚焦条件的优化,建立起四倍体刺槐枝段韧皮部蛋白质双向电泳体系。研究结果表明:采用TCA/丙酮法提取四倍体刺槐枝段韧皮部全蛋白质,选用pH4-7的17 cm IPG胶条,考马斯亮蓝染色上样量550μg,等电聚焦IEF聚焦总伏小时数从60 000 Vh提高到80 000 Vh,并采用12%的分离胶对四倍体刺槐枝段韧皮部全蛋白进行双向电泳,能得到背景清晰、蛋白质点数相对较多,分离度高且重复性好的电泳图谱。利用建立的体系进行双向电泳分离蛋白质,能直接挖点送质谱分析。采用该体系分析四倍体刺槐硬枝扦插生根愈伤组织阶段蛋白质表达差异,共筛选出83个差异蛋白质点,其中上调蛋白15个,新产生蛋白22个,下调蛋白22个,缺失表达24个。     

松材线虫全蛋白的提取及其双向电泳体系优化

目的:一种适用于双向电泳体系的松材线虫全蛋白提取方法的建立及其双向电泳体系的优化.方法:以松材线虫为实验材料,比较2种不同的蛋白提取方法,并对双向电泳中的IPG胶条长度、IPG胶条最适pH范围、上样量等3个方面的条件进行优化.结果:采用TCA-丙酮法提取的蛋白质浓度较高,达到2.18μg/μl.使用pH5 ～8、24cm的IPG干胶条,上样量为120μg,经双向电泳分离可得到背景清晰、分辨率较高的2 - DE图谱,能检测到2 000个左右清晰的蛋白点,含有相对丰富的蛋白信息量.结论:该实验所建立的松材线虫提取方法和优化体系可以为今后松材线虫蛋白质组学的研究奠定技术基础.     

甜瓜叶片中Rubisco的去除及双向电泳体系的优化

为降低Rubisco的干扰,建立适合于甜瓜叶片蛋白质的双向电泳技术,本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明,Mg/NP-40/PEG3350/TCA/丙酮提取法可去除样品中绝大多数的Rubisco,使低丰度蛋白得以检测,适合后续分析。以该法提取的蛋白,采用600、800、1000和l200μg四种上样量进行双向电泳,上样量为l000μg的样品用pH3～10的IPG胶条,在2-DE胶上分辨出质量好、数量多的蛋白质点（562个）。因此,Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法,l000μg是最适上样量。     

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。     

小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体. 在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系. 结果表明,采用20%、24%和40% Percoll密度梯度和28% Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2 mol/L硫脲,4% CHAPS(W/V),65 mmol/L DTT,0.5% IPG缓冲液(V/V),0.001% 溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11% SDS-PAGE分离胶,上样量为160 μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离. 经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.     

沙漠小球藻的蛋白双向电泳分析

为建立适用于小球藻(Chlorellasp.TLD6B)蛋白质组分析的双向电泳体系,该研究比较了TCA/丙酮沉淀法和Trisol提取法对小球藻蛋白的提取效果,不同pH梯度IPG胶条(pH3~10和pH4~7)、不同蛋白质上样量、不同聚焦程序对小球藻蛋白的分离效果。结果表明:(1)采用Trisol提取法可获得较高纯度蛋白,当选择24cm pH 3~10的线性IPG胶条时,上样量为500μg,聚焦80 000Vh效果最佳,可分辨蛋白点达726个;当选择24cm pH 4~7的线性IPG胶条时,上样量为1 000μg,聚焦80 000Vh效果最佳,可分辨蛋白点达1 230个。(2)该实验随机挑选了10个胶内蛋白点进行MALDI-TOF/TOF-MS鉴定分析表明,其中8个蛋白点鉴定成功,进一步说明Trisol提取法可适用于小球藻双向电泳分析。     

多子房小麦蛋白质双向电泳体系的建立及优化

为建立适用于显性多子房小麦细胞质效应的蛋白质双向电泳体系,以显性多子房小麦材料DUOII与特异细胞质材料TeZhiI杂交的F1幼穗为材料,采用TCA-丙酮法提取蛋白质,并在IPG胶条长度和pH范围、SDS-PAGE凝胶浓度及蛋白质上样量等方面,对多子房小麦幼穗蛋白质双向电泳体系进行了探究与优化.结果表明,本文采用的蛋白质定量方法准确度高(R²=0.9999),确立了17 cm, pH4～7的IPG胶条, 12% SDS-PAGE分离胶,上样量为900 μg的双向电泳方法体系,获得了最适合本研究蛋白质组分析的双向电泳图谱. 经PDQuest 2DE 8.0.1软件分析,2-DE图谱上可分辨出1.444±14个清晰蛋白质点,且重复性较高(95%), 相关系数为0.960. 建立了一套适用于显性多子房小麦细胞质效应研究的蛋白质双向电泳体系.     

二维电泳分离牛精子蛋白的技术研究

二维电泳是蛋白质分离技术并可由于对精子蛋白的分离。本研究旨在通过对双向电泳条件的研究摸索出一种适用于分离牛精子蛋白的二维电泳技术,并利用其对牛精子蛋白进行分离鉴定。在实验中,优化了等电聚焦程序,研究了精子蛋白的不同制备方法、不同上样量、不同胶条长度对电泳结果的影响。结果表明,采用尿素－盐酸胍两步裂解法裂解精子细胞制备蛋白,使用13cm非线性胶条进行蛋白二维电泳,能获得较好的电泳图谱。图谱经二维电泳软件分析,可检测出约800多个蛋白质点,分子量基本分布在10~100KD、等电点约为4~9的区域内。对精子蛋白二维电泳条件的摸索,为后续牛精子X、Y差异蛋白的检测和分析奠定了理论基础。     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

双向电泳分析鸢尾绿白嵌合叶片的蛋白质

利用双向聚丙烯酰胺凝胶电泳对鸢尾（Iris japonica)绿白嵌合叶片的蛋白质进行分离,并初步鉴定了蛋白质的相对分子量和等电点。每个电泳图谱共检测到400余个蛋白点,其中至少13个蛋白的表达变化明显;结果表明,嵌合叶片的绿色与白色叶组织具有明显不同的蛋白质双向电泳图谱。与数据库中拟南芥双向电泳图谱相比较,发现Rubisco大亚基,标记为W和T蛋白的表达变化与产生绿白嵌合叶片的表型密切相关。     

Technical updates to basic proteins focalization using IPG strips

ABSTRACT: BACKGROUND: Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. RESULTS: Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6--11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks. CONCLUSION: According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.     

2-D DIGE analysis of the budding yeast pH 6-11 proteome in meiosis

Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis.     

Using standard positions and image fusion to create proteome maps from collections of two-dimensional gel electrophoresis images

Databases for two-dimensional protein gels pose new challenges in extracting meaningful information from large numbers of experiments. In order to create expression profiles, positions of corresponding protein spots across all gel images have to be established. In larger gel sets errors may accumulate rapidly during this spot matching process, effectively limiting the number of samples available for data mining. Here we present a novel approach for organizing spot data based on the concept of a standard position for a protein species. Standard positions are meaningful average positions that are determined using all occurrences of a protein species. They can be extended to spots that are not annotated via interpolation. The standard position of a spot can serve as a unifying index across all gels in a database, thus allowing creation and analysis of expression profiles that span the whole collection. The standard position gives a much more accurate estimation of a spot's position on a gel than can be obtained using theoretical isoelectric point and molecular weight. Positional indexing is a complement to a priori identifications (e.g. by mass spectrometry or Edman degradation). Moreover it can be used in advance to select spots that are worth identifying because they show relevant expression profiles. Furthermore, we show how to combine all spots that occur on any of the gels into one synthetic but nevertheless realistic-looking image. This composite image is produced such that all spots have their standard positions. It can serve as a proteome reference map for an organism. As an application, we have computed a reference map from 23 gel images of Bacillus subtilis, using an enhanced prerelease version of the gel analysis software Delta2D (DECODON, Greifswald, Germany).     

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.