DIGE技术及其在植物蛋白质组学研究中的应用

双向荧光差异凝胶电泳(DIGE)是一项新出现的荧光标记定量蛋白质组学技术,比经典的双向凝胶电泳(2-DE)具有更高的动力学范围和灵敏性。文章综述了DIGE蛋白质组学的基本原理、实验方法、在植物蛋白质组学研究中的应用和局限性,并对DIGE技术的应用做了展望。     

三种花生幼胚蛋白质提取方法比较研究

植物组织(或细胞)的蛋白质提取效率与效果直接影响蛋白质双向凝胶电泳等实验的结果。为探索建立适用于花生幼胚蛋白质(双向凝胶电泳用)提取的最佳条件,尝试了磷酸缓冲液直接提取法、改良的荔枝胚胎蛋白提取法和Trizol(附加)提取法等3种提取方法,根据蛋白提取得率、试剂成本、双向电泳图谱的质量(蛋白质斑点的丰度、分布特点)进行初步评价。结果表明,磷酸缓冲液直接提取法简单但总体效果较差,改良的荔枝胚胎蛋白提取法综合评价最好,与双向凝胶电泳条件更兼容。     

双向凝胶电泳图谱用于常见尸食性蝇类初孵幼虫的鉴别

双向凝胶电泳分析技术已在生物科学各领域被广泛应用,蛋白质组作图的意义已经日益显现。通过对4种常见尸食性蝇类初孵幼虫蛋白质组双向凝胶电泳和图象分析,发现各种类间双向凝胶电泳图谱差异显著,并对相应的等电点和相对分子量进行聚类和判别分析,结果表明,建立合适的尸食性蝇类初孵幼虫的双向凝胶电泳图谱可用于鉴别形态学极易混淆的昆虫种类。图4参17     

浅析双向凝胶电泳的样品制备

对于双向凝胶电泳实验来说最重要的环节就是样品制备,样品的好坏直接关系到整个实验最终的成败。本文将对样品制备进行简要的阐述,并以大肠杆菌为例介绍具体的制备步骤。     

蛋白质组技术进展

蛋白质组研究是近年兴起的生命科学的前沿领域,是生命科学进入后基因组时代的标志之一。蛋白质组研究中的主要技术是双向凝胶电泳技术和质谱技术。本文简要综述了双向凝胶电泳和质谱技术的现状及存在的问题。     

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.     

蛋白质定量

在双向凝胶电泳实验中蛋白质样品的定量是十分重要的步骤,直接关系到结果的准确性及重复性,目前有多种方法可以用于蛋白质样品浓度的测定,针对不同的样品以及不同的实验需要选择行当的方法,本文对常见三种定量方法进行了介绍。     

鼻咽癌原代培养细胞分泌性蛋白图谱的初步研究

目的:建立细胞分泌性蛋白的蛋白质组学研究方法,在此基础上初步建立鼻咽癌细胞分泌蛋白图谱。方法:通过组织块法进行鼻咽癌细胞原代培养,收集细胞上清,超滤法脱盐并浓缩上清蛋白质,双向凝胶电泳技术建立细胞分泌性蛋白图谱。结果:鼻咽癌细胞原代培养取得成功,并初步建立了鼻咽癌细胞分泌性蛋白的双向凝胶电泳图谱。结论:建立了细胞分泌性蛋白的蛋白质组学研究方法,开展了鼻咽癌细胞分泌组学研究,为阐述鼻咽癌癌变机制提供了依据,也为后续研究打下了坚实的实验基础。     

双向凝胶电泳缺陷胶及其原因

双向凝胶电泳是蛋白质组学研究中的关键技术之一,涉及较多的实验步骤和试剂,常出现缺陷胶而导致实验失败。从数千张电泳胶图中选取了21张典型的缺陷胶图,对它们进行了归类和总结,分析和讨论了形成缺陷胶的多种原因,提出了实验改进的方法和注意事项。     

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。     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Comparative proteomics and difference gel electrophoresis

The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.     

Photocontrol of the polypeptide composition ofEuglena

Planta - Two-dimensional gel electrophoresis resolved total cellular protein fromEuglena gracilis Klebs var.bacillaris Cori into 650 polypeptides detectable by silver staining. Exposure of...     

Two-dimensional analysis of gliadin proteins associated with quality in durum wheat: chromosomal location of genes for their synthesis

Theoretical and Applied Genetics - Two-dimensional electrophoresis was used to fractionate the gliadin proteins from the endosperm of durum wheat. The increased resolution of the system, as...     

Proteomics: quantitative and physical mapping of cellular proteins

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level. Proteomics can be divided into expression proteomics, the study of global changes in protein expression, and cell-map proteomics, the systematic study of protein-protein interactions through the isolation of protein complexes.     

Electrophoresis of honey: characterization of trace proteins from a complex biological matrix by silver staining

The proteins of unconcentrated honey have been detected with the methylamine-incorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high-resolution two-dimensional electrophoresis. The former consistently reveals at least 19 protein bands in a variety of Australian honeys. Two-dimensional electrophoresis gave patterns of poor resolution but proved useful for further characterization of the major protein constituents. The protein patterns were unaffected by centrifugation of the samples prior to preparation for electrophoresis.     

Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed.     

Ribosomal Changes during Induction of Cold Hardiness in Black Locust Seedlings

Protein synthesis has been implicated in the cold-hardening process. Ribosomes from cold hardy and nonhardy black locust (Robinia pseudoacacia L.) seedlings were compared to determine if cold acclimation is related to alteration of ribosomal structure. Ribosomal structure, as indicated by thermal melting profiles, appears to be altered during induction of hardiness. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicates at least 17 proteins from hardy seedlings that are different from those of nonhardy seedlings. These different proteins may be partially responsible for the different thermal melting profiles observed.     

Horizontal two-dimensional electrophoresis of plasma butyrylcholinesterase

Genetic variants of human plasma butyrylcholinesterase have been characterized and are highly relevant to anesthesiology. They might also represent potential genetic markers for neuropsychiatric disorders. Two-dimensional electrophoresis with isoelectrofocusing in the first and polyacrylamide gel electrophoresis in the second dimension has proved to be a powerful tool in search for genetic variants. Butyrylcholinesterase is an oligomeric enzyme with considerable charge heterogeneity. Conventional two-dimensional electrophoresis proved unsuitable for this enzyme possibly due to its tendency to aggregate by hydrophobic interactions. The inversion of the sequence applying polyacrylamide gel electrophoresis in the first and isoelectric focusing in the second dimension circumvented this problem.     

Discrimination between RNA circles, interlocked RNA circles and lariats using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis can be used to identify structural forms of RNA such as linear RNA, circular RNA, interlocked circles and lariats. The procedure is based upon the characteristic migration behaviour of the degradation products derived from the intact structures present already before the start of the experiment or formed during or after electrophoresis in the first dimension. After autoradiography to detect the positions of the radiolabeled RNA molecules, circles broken during electrophoresis of the first dimension give rise to horizontal lines touching the diagonal formed by linear RNAs at a point corresponding to the length of the RNA circle from which it was derived. Products derived from interlocked RNA circles by breakage after completion of the first dimension appear on a vertical line underneath the intact complex and consist of free RNA circles and their linear derivatives. Broken lariats give rise to two lines depending on the location of the break. Lariats with broken tails are present on a line to a position that corresponds to the length of their tail and that runs parallel to the diagonal formed by linear products. Lariats with a broken eye form a line running from the position of the intact product to the diagonal formed by the linear RNAs.