电泳,凝胶,双向

摘要： 蛋白质组学是对基因编码蛋白质进行大规模分析的一门新兴学科.本文概述蛋白质组学的基本技术:双相凝胶电泳、多维液相色谱、生物质谱技术及蛋白质芯片在2型糖尿病(T2DM)中的应用,从而探讨T2DM中与炎症及脂代谢紊乱相关的生物蛋白:C反应蛋白、α2-巨球蛋白、载脂蛋白A1、Clusterin及锌-α-2糖蛋白的研究进展,发现早期诊断T2DM新的特异性标志物及药物作用靶点.

摘要： Objective To screen altered proteins of hippocampus in the stress-induced depression (STRID) rat model, and explore the potential molecular mechanism. Methods Twenty Sprague-Dawley rats were randomly divided into the control group and STRID group, 10 rats in each group. Chronic unpredictable mild stress (CUMS) methods including fasting for solids and liquids, electric foot-shock, reversing day and night, cold water swimming, cage tilt, scare stimulation and tail pinch were conducted on STRID rats with no repeats for 28 days to make up the depression animal model. The control group was normally fed during this period. After the stress stimulation, the hippocampus protein samples were used for two dimensional electrophoresis to screen the differentially expressed protein, and then mass spectrum identification and function analyze were conducted. Results Compared with the control group, 34 proteins were altered in STRID group. Among which, 18 were up-regulated, and 16 were down-regulated. The differentially expressed proteins mainly located in cytoplasm, mitochondrion, extracellular exosome and myelin sheath. The involved signaling pathways included metabolic pathway, oxidative phosphorylation pathway, and Alzheimer's disease, Parkinson's disease and Huntington's disease pathways. Conclusion The altered proteins and dysfunction of nerve signaling, and the excess of oxidative phosphorylation in hippocampus of STRID rats may be one of the pathogenesises.%目的 筛选应激性抑郁症(STRID)模型大鼠海马组织中的差异表达蛋白,初步探讨STRID的发病机制.方法 将20只成年SD大鼠随机分为对照组和STRID组,每组10只.STRID组给予慢性温和不可预知应激(CUMS)干预(包括禁食水、电刺激足底、颠倒昼夜、冰水游泳、倾斜鼠笼、惊吓刺激、夹尾等方法)连续不重复刺激28 d建模,对照组在此期间正常喂养.在STRID模型建立完成后提取2组大鼠海马组织进行蛋白双向电泳,筛选出差异表达蛋白点后进行质谱鉴定和蛋白功能分析.结果 2组共鉴定出34个差异表达蛋白,其中STRID组较对照组表达上调18个、下调16个.差异表达蛋白主要集中于细胞质、线粒体、细胞外泌体和髓鞘中,主要参与代谢信号通路、氧化磷酸化信号通路,以及阿尔茨海默症、帕金森病和亨廷顿症信号通路.结论 STRID大鼠海马组织存在蛋白的异常表达和神经信号传导异常,过度的氧化磷酸化可能是其致病机制之一.

摘要： 目的：分析缺失编码Ⅲ型分泌系统的大质粒pCD1对鼠疫耶尔森菌蛋白质表达谱的影响，探索鼠疫菌的潜在致病机制，为鼠疫疫苗研制提供新线索。方法通过双向电泳与质谱分析相结合的方法，比较鼠疫菌在26°C和37°C不同培养温度下，全菌蛋白表达谱差异，鉴定差异蛋白并对其功能进行初步分析。结果成功分离并鉴定了鼠疫菌201株和201△pCD1缺失突变株中的差异蛋白，其中26°C条件下鉴定到24个差异蛋白，37°C条件下鉴定到25个差异蛋白，内含7个由pCD1质粒编码的蛋白。结论通过比较蛋白质组学研究，发现大质粒pCD1缺失后，多种染色体编码蛋白的丰度发生了显著变化，暗示大质粒能调控染色体编码基因的表达。

摘要： Objective To explore the potential pathogenesis of Yersinia pestis and provide new clues for vaccine development through comparative proteomic analysis of wild-type and pCD1 cured strain of Yersinia pestis 201.Methods Differentially expressed proteins at 26°C and 37°C were separated and identified using two-dimensional electrophoresis coupled with mass spectrometry .Results A total of 24 differently expressed proteins were successfully identified from the samples of bacteria grown at 26°C and 25 proteins at 37°C.Among these, 7 proteins were encoded by pCD 1 plasmid. Conclusion Through comparative proteomic research, we have found that the abundance of several proteins can be dramatically changed when the large plasmid pCD 1 is missing,suggesting that the plasmid can regulate the expression of many genes located in the chromosome .%目的：分析缺失编码Ⅲ型分泌系统的大质粒pCD1对鼠疫耶尔森菌蛋白质表达谱的影响，探索鼠疫菌的潜在致病机制，为鼠疫疫苗研制提供新线索。方法通过双向电泳与质谱分析相结合的方法，比较鼠疫菌在26°C和37°C不同培养温度下，全菌蛋白表达谱差异，鉴定差异蛋白并对其功能进行初步分析。结果成功分离并鉴定了鼠疫菌201株和201△pCD1缺失突变株中的差异蛋白，其中26°C条件下鉴定到24个差异蛋白，37°C条件下鉴定到25个差异蛋白，内含7个由pCD1质粒编码的蛋白。结论通过比较蛋白质组学研究，发现大质粒pCD1缺失后，多种染色体编码蛋白的丰度发生了显著变化，暗示大质粒能调控染色体编码基因的表达。

摘要： Objective To study the differential expression of sperm proteins between fertile men and infertile men with varicocele(VC). Methods Sperm samples were analyzed by computer-assisted semen analysis system. Sperm proteins were extracted by percoll density gradient centrifugation, and the separation of total proteins was performed by two-dimensional electrophoresis(2-DE). The differentially expressed proteins of two groups were analyzed with PDQuest software. Results The sperm motility in infertile men with VC demonstrated a significant decrease compared with the fertile men(P0.05). Protein profile of sperm was acquired with clear background, high resolution and better reproduction.Total of 36 different proteins were found, including 11 upregulated protein and 19 downregulated proteins in infertile men with VC group, and two proteins only expressed in infertile men with VC group, the other four proteins only expressed in fertile men group. Conclusion There are differential expression of sperm proteins between fertile men and infertile men with VC. Differential proteins analysis will be helpful to explore the molecular mechanism of VC-associated male infertility.%目的：比较分析正常生育男性与精索静脉曲张（varicocele, VC）不育患者差异表达的精子蛋白质。方法应用计算机辅助精子分析系统行精液分析，采取Percoll密度梯度离心法提取正常生育男性与VC不育患者精子，利用双向电泳（two-dimensional electrophoresis,2-DE）分离精子蛋白质，建立并优化精子蛋白质2-DE图谱，利用PDQuest8.0软件分析两组蛋白质的差异表达。结果正常生育组精子活力高于VC不育组，两组比较差异有统计学意义（P＜0.05），两组精子密度比较差异无统计学意义（P＞0.05）。获得了分辨率和重复性均较好的2-DE图谱，两组差异点共36个，VC不育组有11个蛋白位点表达上调，19个蛋白位点表达下调，2个在VC不育组特异性表达，4个在正常生育组特异性表达。结论 VC不育患者与正常生育男性的精子蛋白存在差异，这些差异蛋白质的分离为深入探讨VC导致不育的分子机制奠定了基础。

摘要： 目的 了解毛乳头细胞在凝集生长状态下分泌性蛋白组的表达.方法 以凝集和非凝集生长的毛乳头细胞为研究对象,分别制备分泌性蛋白质,以双向凝胶电泳技术和PDQuest软件分析二者之间的蛋白图谱的差异,通过基质辅助激光解吸电离飞行时间串联质谱(MLDI-TOF-MS)鉴定表达差异的蛋白质,通过蛋白质数据库NCBInr检索分析.结果 建立了重复性好、分辨率高的双向电泳图谱;在凝集生长和非凝集生长的毛乳头细胞分泌性蛋白质中分别检测到(1 134±52)个和(1 078±36)个蛋白点,大多匹配.按照差异量在5倍以上的标准,二者存在差异蛋白质28个,经过MLDI-TOF-MS质谱鉴定出10种差异表达蛋白点,其中8种蛋白质表达上调,分别为RhoGDP分离抑制剂1、细丝蛋白A、胱抑素C、纤维连接蛋白、亲环素A、前胶原C端蛋白酶增强子、组织金属蛋白酶抑制剂、组织金属蛋白酶-2抑制剂.2种蛋白质表达下调,为神经肽h3和基质金属蛋白酶-3金属蛋白酶组织抑制剂-1复合物复合物.结论 凝集生长和非凝集生长毛乳头细胞差异蛋白主要涉及信号通路、细胞增殖与分化、细胞外基质合成及降解等功能.%Objective To study the expression ot secreted proteins in aggregated dermal papilla cells (DPCs).Methods DPCs were isolated from human scalp tissue and subjected to primary culture and subculture.Aggregated and non-aggregated DPCs served as the subject of this study.Secreted proteins were prepared from these cells and subjected to two-dimensional polyacrylamide gel electrophoresis.Differentially expressed proteins were screened by the PDQuest image analysis software.Protein spots were digested and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry,and finally analyzed using the National Center for Biotechnology Information (NCBI) non-redundant (Nr) protein database.Results Two-dimensional electrophoresis maps with good repeatability and high resolution were established.Image analysis of 2-D gels revealed that the average number of detected protein spots was 1 134 ± 52 and 1 078 ± 36 in aggregated and nonaggregated DPCs respectively,and the majority of these protein spots were matched between aggregated and nonaggregated DPCs.Twenty-eight protein spots showed more than 5-fold difference between the two groups of cells,and 10 proteins were preliminarily identified as differentially expressed proteins by peptide-mass fingerprinting.Of these differentially expressed proteins,8 proteins including Rhogdi 1,filamin A,cystatin C,fibronectin,cyclophilin A,procollagen C proteinase enhancer 1,tissue inhibitor of metalloproteinase and tissue inhibitor of metalloproteinase-2 were up-regulated,and 2 proteins including neuropolypeptide h3 and matrix metalloproteinase-3/tissue inhibitor of metalloproteinase-1 complex were down-regulated in aggregated DPCs compared with non-aggregated DPCs.Conclusions Differentially expressed proteins between aggregated and non-aggregated DPCs are mainly implicated in cell signaling pathway,cellular proliferation and differentiation,extracellular matrix synthesis and degradation,and so on.

摘要： 目的：初步探讨phoN1基因在志贺菌属中的功能。方法使用λ-Red重组系统构建弗氏志贺菌2a 301株phoN1缺失突变株，通过比较蛋白质组学方法研究突变株与野生株的蛋白表达谱差异，并利用豚鼠角膜炎模型、HeLa细胞竞争侵袭实验评价两者之间的毒力差异。结果成功构建了phoN1缺失突变株，蛋白质学研究发现， phoN1缺失对其他胞内蛋白无显著影响，豚鼠角膜实验和HeLa细胞竞争侵袭实验证实，phoN1缺失不影响志贺菌属的侵袭能力。结论 phoN1在体外生存、侵袭宿主细胞过程中未发挥显著作用，其功能有待进一步研究。%Objective To explore the function of gene phoN1 in Shigella flexneri.Method Using the λ-Red recombi-nant system, phoN1was knocked out from S.flexneri 2a strain 301.Comparative proteomics was performed to analyze the differences between mutant and wild-type strains in protein expression profiles .Sereny tests and competitive infection assays were carried out to compare the virulence of mutant and wild-type strains .Results The deletion mutant of phoN1 was suc-cessfully constructed .No significant difference between the two strains was found in the comparative proteomics analyses . The function of gene phoN1 might be unrelated to the invasion ability of S.flexneri according to the results of Sereny tests and competitive infection assays .Conclusion Gene phoN1 might be of no use for the in vitro survival and host cell invasion of S.flexneri.

摘要： Objective To compare the separation effects of protein samples extracted by two different methods with two -dimensional gel electrophoresis (2-DE) .Methods Ultrasonic disruption and glass-beads grounding were used to prepare protein samples of Gram-negative bacteria , Gram-positive bacteria and animal tissues .The actual results of the two sample preparation methods were compared by 2-DE.Results The 2-DE maps of samples extracted by the two methods were obtained.Conclusion The 2-DE maps of glass-beads grounding samples are better than those of ultrasonic disruption thanks to their lower backgrounds , which are beneficial for further image analyses .%目的：比较两种破碎方法提取的蛋白质样品在双向电泳（2-DE）中的分离效果。方法用超声破碎和玻璃珠振荡破碎方法分别制备革兰阴性菌、革兰阳性菌、动物组织的蛋白抽提样品，通过2-DE比较两种制样方法的实际效果。结果获得了革兰阴性菌、革兰阳性菌、动物组织在两种制样条件下的2-DE图。结论通过对比不同样品的电泳结果发现，玻璃珠振荡破碎法制样背景更低，更有利于后续的图像分析。

摘要： 目的比较两种破碎方法提取的蛋白质样品在双向电泳（2-DE）中的分离效果。方法用超声破碎和玻璃珠振荡破碎方法分别制备革兰阴性菌、革兰阳性菌、动物组织的蛋白抽提样品,通过2-DE比较两种制样方法的实际效果。结果获得了革兰阴性菌、革兰阳性菌、动物组织在两种制样条件下的2-DE图。结论通过对比不同样品的电泳结果发现,玻璃珠振荡破碎法制样背景更低,更有利于后续的图像分析。

摘要： 目的 利用比较蛋白质组学方法鉴定胰腺癌早期诊断的差异表达蛋白,筛选出潜在的特异性候选标志物.方法 采用双向差异凝胶电泳(2D-DIGE)分离2007年5月至2008年9月收治的20例胰腺癌患者、10例慢性胰腺炎患者和20名健康体检者外周血清蛋白质,利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术对差异显著的蛋白进行鉴定,并对鉴定出的差异表达蛋白进行Western blot验证.结果 成功鉴定出胰腺癌差异表达蛋白补体3(C3).C3在胰腺癌组织中的灰度比为1.63±0.28,高于正常胰腺的0.65 ±0.13(t=11.81,P=0.00)和慢性胰腺炎的0.88±0.19(t =9.93,P=0.00).结论 应用2D-DIGE联合MALDF-TOF-MS技术筛选胰腺癌患者外周血清中的特异性生物标志物的方法快速、有效.检测鉴定的差异蛋白质C3可能是胰腺癌早期诊断潜在的血清特异性生物标志物.%Objective To identify protein markers for the early diagnosis of pancreatic cancer by a comparative proteomic method.Methods Comparative analysis on the pancreatic peripheral blood protein profiling from 20 pancreatic cancer patients,10 chronic pancreatitis patients and 20 cancer-free controls from May 2007 to September 2008 was carried out by two-dimensional fluorescence electrophoresis (2D-DIGE).Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).The significance difference proteins were confirmed by Western-blot.Results A differentially expressed proteins:complement 3 (C3) was identified.The gray level of C3 in pancreatic cancer tissue,chronic pancreatitis,and normal control group were 1.63 ± 0.28,0.65 ±0.13(t =11.81,P =0.00) and 0.88 ±0.19 (t =9.93,P =0.00),respectively.C3 was high expression in pancreatic cancer group compared with normal control group.The expression of C3 was higher in pancreatic cancer group than in chronic pancreatitis group.The high expression of C3 in pancreatic carcinoma was confirmed by Western blot.Conclusions 2D-DIGE and MALDI-TOF-MS technology is a quick,easy and practical method to screen for specific biomarkers in serum of patients with pancreatic carcinoma.The identified protein C3 in this study may be as specific serum biomarkers of pancreatic carcinoma.

摘要： 本发明为蛋白质的胶内检测和定量提供了一种新的仪器设计。这种新平台称为累积的时间分辨发射双向凝胶电泳(CuTEDGE)，利用荧光寿命成像的差异来区分来自特异性蛋白质标记的荧光和非特异性背景荧光，致使与现有技术相比在灵敏度和动态范围方面均有明显的改进。该平台主要用于双向凝胶电泳的图像获取，但也用于单向凝胶系统的蛋白质检测以及电转移到例如PVDF膜上的蛋白质。考虑到与现有方法相比，在检测和动态范围方面增加了高达5-6个数量级，所描述的发明在低丰度细胞蛋白质的检测中代表了技术的飞跃，这种技术的飞跃是生物标志物发现的领域中迫切需要的。

摘要： 本发明公开了一种植物质膜蛋白再溶解的方法及其在双向荧光差异凝胶电泳中的应用。本发明提供的植物质膜蛋白再溶解的方法，包括如下步骤：将植物质膜蛋白加入裂解液中，在冰浴中超声处理，然后涡混使所述植物质膜蛋白溶解，最后离心收集上清液，即为植物质膜蛋白溶液；所述裂解液是在DIGE裂解缓冲液中加入N-十二烷基-β-D-麦芽糖苷并使其浓度为0.5-2g/100mL得到的。植物质膜蛋白的丰度低，疏水性强，其提取及再溶解存在着一定的难度，给质膜蛋白质组学的研究带来了极大的挑战。采用本发明提供的再溶解方法，植物质膜蛋白的提取效率增加了近两倍，进行2-DDIGE后能够得到重复性好，分辨率高的电泳结果比现有方法显示更多的蛋白点。

摘要： 本发明公开了一种重金属胁迫下水稻总蛋白的双向凝胶电泳分离方法，包括：(a)提取水稻根蛋白粗品，用乙醚和乙醇的混合溶液洗涤，冷冻干燥后溶于裂解液，水浴加热0.5～2小时，离心，取上清液用丙酮沉淀，离心分离沉淀，冷冻干燥；(b)将沉淀溶于增溶液中，用冷丙酮于-20～-15°C沉淀，离心分离沉淀，重复该步骤2～4次后，将沉淀冷冻干燥；(c)将步骤(b)获得的沉淀溶于增溶液中，利用双向凝胶电泳分离。本发明方法通过增加尿素和硫脲等不同强度的表面去垢剂和多种离液剂，再通过添加PVP和乙醇乙醚的洗涤来去除其它杂质，最后还通过分步溶解来获得更纯的蛋白，同时降低蛋白样品的复杂性，得到了高纯度的水稻根总蛋白。

摘要： 本发明公开了一种结合双向凝胶电泳和质谱鉴定糖蛋白糖链结构的方法。所述方法包括以下步骤：S1、对生物样本中的糖蛋白进行特异性富集并通过双向凝胶电泳进行分离，根据目标糖蛋白的分子量和等电点选定目标糖蛋白异构体凝胶斑点；S2、使用双向凝胶电泳蛋白印迹和质谱对选定的目标糖蛋白异构体凝胶斑点进行确认；S3、对步骤S2中确认了的目标糖蛋白异构体凝胶斑点进行胶内酶解和质谱分析，鉴定其肽段序列和糖链结构。本发明适用于各种蛋白翻译后修饰(糖基化，泛素化和乙酰化等)生物标志物异构体和修饰体的分离以及结构鉴定，可以显著提高翻译后修饰蛋白作为诊断标志物的灵敏度和特异性。

摘要： 本发明公开了一种筛选耐盐碱性植物材料的双向电泳凝胶考马斯亮蓝染色和脱色方法，所述方法包括如下步骤：步骤一、配制含有考马斯亮蓝R250、甲醇、乙酸、蒸馏水定容的染色液，混匀后静置0.5～1.5小时，过滤后室温保存备用。步骤二、配制含有乙醇、乙酸、蒸馏水定容的脱色液。步骤三、双向SDS凝胶蒸馏水冲洗2～3次，放入染色盒中，加入染色液，利用保鲜膜封口，室温条件下，摇床上染色2～3小时。步骤四、脱色：倒出染色液，蒸馏水冲洗凝胶2～4次；加入脱色液，脱色液用量为染色液用量的1～2倍，室温条件下，摇床上脱色2～3小时，蒸馏水冲洗2～4次。本发明在常规技术方法上进一步优化，省略掉前固定等程序，染色和脱色速度快、效果好。

摘要： 本发明提出了一种木薯蛋白质双向电泳凝胶，原料配方中，30％丙烯酰胺溶液和40％丙烯酰胺溶液混合使用。本发明中的用于木薯蛋白质双向电泳的凝胶，只改变丙烯酰胺的混合方式，操作简单，等同条件下可提高凝胶的分辨率，可重复率高，符合实验要求。

摘要： 本发明公开了种植物质膜蛋白再溶解的方法及其在双向荧光差异凝胶电泳中的应用。本发明提供的植物质膜蛋白再溶解的方法，包括如下步骤：将植物质膜蛋白加入裂解液中，在冰浴中超声处理，然后涡混使所述植物质膜蛋白溶解，最后离心收集上清液，即为植物质膜蛋白溶液；所述裂解液是在DIGE裂解缓冲液中加入N-十二烷基-β-D-麦芽糖苷并使其浓度为0.5-2g/100mL得到的。植物质膜蛋白的丰度低，疏水性强，其提取及再溶解存在着一定的难度，给质膜蛋白质组学的研究带来了极大的挑战。采用本发明提供的再溶解方法，植物质膜蛋白的提取效率增加了近两倍，进行2-DDIGE后能够得到重复性好，分辨率高的电泳结果比现有方法显示更多的蛋白点。

摘要： 本发明为蛋白质的胶内检测和定量提供了一种新的仪器设计。这种新平台称为累积的时间分辨发射双向凝胶电泳(CuTEDGE)，利用荧光寿命成像的差异来区分来自特异性蛋白质标记的荧光和非特异性背景荧光，致使与现有技术相比在灵敏度和动态范围方面均有明显的改进。该平台主要用于双向凝胶电泳的图像获取，但也用于单向凝胶系统的蛋白质检测以及电转移到例如PVDF膜上的蛋白质。考虑到与现有方法相比，在检测和动态范围方面增加了高达5-6个数量级，所描述的发明在低丰度细胞蛋白质的检测中代表了技术的飞跃，这种技术的飞跃是生物标志物发现的领域中迫切需要的。

摘要： 本发明公开了一种重金属胁迫下水稻总蛋白的双向凝胶电泳分离方法，包括：(a)提取水稻根蛋白粗品，用乙醚和乙醇的混合溶液洗涤，冷冻干燥后溶于裂解液，水浴加热0.5～2小时，离心，取上清液用丙酮沉淀，离心分离沉淀，冷冻干燥；(b)将沉淀溶于增溶液中，用冷丙酮于-20～-15°C沉淀，离心分离沉淀，重复该步骤2～4次后，将沉淀冷冻干燥；(c)将步骤(b)获得的沉淀溶于增溶液中，利用双向凝胶电泳分离。本发明方法通过增加尿素和硫脲等不同强度的表面去垢剂和多种离液剂，再通过添加PVP和乙醇乙醚的洗涤来去除其它杂质，最后还通过分步溶解来获得更纯的蛋白，同时降低蛋白样品的复杂性，得到了高纯度的水稻根总蛋白。

摘要： 双向平板凝胶电泳装置，包括上下槽。上槽含两侧壁、底板构成的H形支架，两侧壁设圆孔和电极插板槽道。上槽设前、后壁，前壁是上、下边均设凹口的平板。后壁是矩形平板。本发明含一块可叠置于上槽平行双凹平板，两者几何形状、面积相同的双凹平板，后者左右垂直边放置间条，上覆玻板，用夹固定。本发明另含一块可叠置于上槽平行双凹平板的垂直双凹平板，后者两凹口呈垂直走向；后者垂直边上的凹口，嵌一短间条。左右垂直边放置常规长间条，上覆玻璃板，用夹固定。本发明含又一块可叠置于上槽平行双凹平板的开三凹口平板；该三凹平板两垂直边凹口，用间条嵌补，左右边放置常规间条，上覆玻板，夹定。