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研究了植物凤凰木种子中的蛋白质成分,试图分离出新的核糖体失活蛋白,凤凰木种子经磷酸盐缓冲液抽提,硫酸铵分级盐析,分子筛和阴离子交换剂等多次柱层析,分离出三种组分DrI,DrⅡ和DrⅢ.SDS-PAGE和IEF实验表明这三种蛋白质均达到单一纯,而HPLC实验则表明它们的纯度不低于90%,它们的分子量据SDS-PAGE和HPLC实验分别约23500、26000、285蛋0.IEF实验测得三者的等电点均 相似文献
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大熊猫子宫钙调素的分离纯化和性质的研究 总被引:2,自引:0,他引:2
以大熊猫子宫为材料分离纯化了钙调素,经SDS-PAGE,PAGE和等电聚焦电泳鉴定,表现均一。分子量为18800道尔顿,等电点为3.6。该蛋白质分子的N-末端为封闭的。大熊猫子宫钙调素具有其它来源钙调素所特有的一些性质。对环核苷酸磷酸二酯酶有明显的激活作用,还发现对超氧化物歧化酶也有一定激活作用。电泳行为受Ca^2+影响而出现特征性电泳改变,在含有Ca^2+的SDS凝胶电泳中,电泳速度比EGTA存 相似文献
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应用亲和层析法是纯鸡马立克氏病病毒PP38基因重组产物 总被引:3,自引:0,他引:3
利用鸡马立氏病病毒(MDV)I型特异单克隆抗体H19致敏Sepharose4B-CNBr,从感染重组病毒BP38Ⅱ的昆虫细胞中提纯鸡马立克氏病毒PP38基因重组产物,获得良好效果,提纯的蛋白质的SDS-PAGE中表现出一条分子量约为38kDa的蛋白质条带,在免疫印迹试验中该蛋白质带也能被单克隆抗体H19识别。利用该提纯的重组PP38免疫小鼠,所制备的小鼠抗血清在免疫荧光染色试验中不仅能与感染重组病 相似文献
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地衣芽孢杆菌1Baciuus Licheniformis)BL-306产生的胞外β-甘露聚糖酶经硫酸铵分级盐析,DEAE-纤维素柱层析。Sephadex-G100柱凝胶过滤和DEAE-纤维素柱再层析分离纯化,得到SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)均一样品。用SDS-PAGE测得纯化后β-甘露聚糖酶分子量为26000道尔顿。用凝胶等电聚焦电泳(PAGEIEF)测得等电点PI为5.0。该酶 相似文献
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研究了植物凤凰木种子中的蛋白质成分,试图分离出新的核糖体失活蛋白凤凰木种子经磷酸盐缓冲液抽提,硫酸铵分级盐析,分子筛和阴离子交换剂等多次柱层析,分离出三种组分DrⅠ、DrⅡ和DrⅢ.SDS-PAGE和IEF实验表明这三种蛋白质均达到单一纯,而HPLC实验则表明它们的纯度不低于90%。它们的分子量据SDS-PAGE和HPLC实验分别约23500、26000、28500.IEF实验测得三者的等电点均为5.2左右。测定了组分DrⅠ的蛙卵泡裂解活性,其毒性约为天花粉蛋白的1/40.分析了它们的氨基酸组成。估算了三种蛋白质的二级结构含量,结果表明三者均富含β折叠结构。已有的实验事实表明这三种蛋白质的生化行为明显异于核糖体失活蛋白,因而很可能不属于这一蛋白质家族。 相似文献
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苦瓜籽核糖体失活蛋白的理化性质及生物活性 总被引:13,自引:0,他引:13
采用硫酸铵分级分离,假配基亲和层析和SephacrylS-100分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白(RIP).经SDS-PAGE、PAGE、IEF和PAS方法分析均表明为单一蛋白着色带或单一糖蛋白着色带.根据SDS-PAGE和Sephadex G-150分子筛层析结果计算其相对分子质量为3.0×104,经IEF-PAGE结果计算其pI为8.9~9.0.对无细胞系统中蛋白质生物合成抑制活性明显,其IC50为5.3×10- 10 m ol/L左右.体外生物活性试验结果表明其对人肝癌细胞、Vero、SP2/0、3T3、Kb、Navana 等肿瘤细胞株均表现有不同程度的抑制作用.而对完整细胞人胚肺二倍体细胞却毒性极小.因此,上述实验结果为该RIP的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础. 相似文献
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地衣芽孢杆菌β—甘露聚糖酶的纯化及酶学性质 总被引:9,自引:1,他引:9
地衣芽孢杆菌(Bacillus licheniformis)NK-27菌株发酵产生的β-甘露聚糖酶(β-mannanase)经硫酸铵盐析沉淀,两次DEAE纤维素和Sephadex G-100离子交换柱层析以及制备PAGE等步骤,获得了凝胶电泳均一的样品。用SDS-凝胶电泳测得纯化后的β-甘露聚糖酶分子量为26kD,用凝胶聚焦电泳测得等电点P1为5.0。酶反应的最适pH为9.0,最适温度为60℃,稳 相似文献
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尖吻蝮蛇蛇毒出血毒素的纯化与部分性质 总被引:1,自引:0,他引:1
目的 被尖吻蝮蛇 ( Dienagkistrodon acutus) 咬伤会引起严重的出血, 对蛇毒出血毒素的研究有利于治疗蛇伤出血药物筛选。 方法 采用 Sephadex G75, D E A E Sephadex A50, Sephadex G200 和两次 P B E 聚焦层析纯化。 S D S P A G E 电泳和等电聚焦电泳测定纯化样品的纯度和等电点。氨基酸组成用自动氨基酸分析仪测定。以小鼠背部皮下注射部位出血斑的面积来确定最小出血剂量和常规的方法测定酶活性。结果 从尖吻蝮蛇毒中纯化到一个相对分子量为56 000 的出血毒素 ( Da H T3), 经氨基酸组成测定计算,它由 487 个氨基酸残基组成。此成分在 S D S P A G E上显示出一条均一的蛋白染色带, 其p I为550。该出血成分的最小出血剂量是 26μg, 具有蛋白水解酶活力, 其活力为 368, 但没有精氨酯酶和磷脂酶 A2 活力。当加入 E D T A 螯合剂去除金属离子后, 它们的出血活力和蛋白水解酶活力均丧失。 结论 这是从大陆尖吻蝮蛇毒中获得的一个新的出血金属蛋白酶 ( Da H T3)。 相似文献
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皖南尖吻蝮蛇毒出血毒素IV的纯化与初步鉴定 总被引:3,自引:0,他引:3
从皖南尖吻蝮蛇毒液中经DEAE-Sepharose和Sephacryl S-200两步凝胶柱层析首次纯化出一种中分子量血毒素(简称AaHⅣ)。经SDS-PAGE和等电聚焦凝胶电泳测定其分子量为44kD,等电点为pH5.0。从500mg粗毒中可获得20mg AaHⅣ纯品。AaHⅣ有较强的出血活性,最小出血剂量(MHD)为0.4μg。 相似文献
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目的分离、鉴定MPTP诱导慢性帕金森病模型小鼠纹状体差异表达的蛋白质,对MPTP慢性PD动物模型的特异性蛋白质组进行初步探讨,为PD的发病机制提供一定的蛋白质组学依据。方法成功建立MPTP诱导慢性帕金森病小鼠模型,提取模型组和对照组小鼠脑纹状体蛋白质,分别以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行2-DE。图像分析软件PDQUEST8.0分析电泳图谱找出差异表达蛋白,运用MALDI-TOF MS质谱鉴定;其肽质量指纹图(PMF)经MS Fit检索。结果比较MPTP诱导慢性PD模型小鼠和正常对照小鼠纹状体二向电泳图,发现12个蛋白表达异常,最终鉴定出其中4个蛋白质:线粒体裂殖调节因子1(mitochondrial fission regulator 1)、类泛素样蛋白3前体(ubiquitin-like protein 3 precursor)表达下调;S100蛋白A10(proteinS100-A10)、Lin-7 homolog B为新出现点。结论初步鉴定出MPTP慢性PD模型小鼠纹状体部分差异表达蛋白,所发现4个表达异常的蛋白质与帕金森病线粒体的损伤和兴奋性神经毒性密切相关,与PD的发病机制相符,为深入研究帕金森病病理机制奠定了基础。 相似文献
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叶景秀 《中国生物化学与分子生物学报》2012,28(11):1049-1056
春小麦是青海省的主要粮食作物,青海高原干旱频繁且严重,尤以春旱为首,对小麦生长发育造成严重影响.为了从蛋白质组学水平分析小麦对干旱胁迫的应答特征,探讨小麦可能的抗旱机制,本研究对青海主栽春小麦品种青春38幼苗进行聚乙二醇 (polyethylene glycol,PEG) 6000胁迫和复水处理,采用IEF/SDS PAGE双向凝胶电泳技术,对PEG胁迫和复水处理的小麦叶片总蛋白质分别与正常浇水的对照进行差异蛋白质组学研究,经考马斯亮蓝G 250染色获得清晰度和重复性较好的双向电泳图谱.PD Quest软件处理分析各对照和处理图谱,在等电点4.0~7.0线性范围内,均可识别650个以上清晰蛋白质点,获得比较明显的差异表达蛋白点43个,其中8个蛋白点重复.从35个差异蛋白点中选取24个差异点进行MALDI-TOF-TOF-MS肽质量指纹图谱分析,应用Mascot软件在NCBInr数据库中搜索鉴定蛋白质,得到22个阳性结果.对鉴定得到的差异表达蛋白进行功能分析,它们分别参与了光合作用、蛋白质合成、能量代谢途径、细胞防御、氧化还原、运输、信号转导等过程,而且根据差异表达蛋白功能分类所占比例,发现干旱胁迫与光合作用关系最为紧密. 相似文献
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红莲型水稻细胞质雄性不育花粉总蛋白质初步比较分析 总被引:4,自引:0,他引:4
采用固相pH梯度/SDS-PAGE双向电泳对红莲型细胞质雄性不育水稻的不育系(YTA)和保持系(YTB)二核期花粉总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。用PDQuest2DE软件可识别约1500个蛋白质点,其中差异表达的蛋白质点数为120。将其中15个差异点采用基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption/ionizaton time of flight mass spectrometry,MALDI-TOF-MS)进行了肽质指纹图分析,通过采用Mascot软件对MSDB数据库查询,其中7个蛋白质点得到了鉴定。YTA相对于YTB有部分参与物质和能量代谢的蛋白质缺失或表达量降低,这些蛋白质分别是水稻线粒体H -转运ATPase(H -ATPase)α链、盐诱导型膜联蛋白、线粒体NAD -依赖型苹果酶和磷酸核糖焦磷酸合成酶等。这些蛋白质的表达下调或缺失可能与线粒体提供能量不足而导致的花粉不能正常发育有关。线粒体电压依赖性阴离子通道(VDAC)这一重要蛋白质在YTA中的上调表达有可能与花粉败育过程中细胞的程序性死亡相关。 相似文献
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Koay SY Gam LH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(22):2179-2183
Orthosiphon aristatus is a traditionally used medicinal plant. In order to study the proteome of the plant, we have developed a simple plant protein extraction method by direct extraction of protein using a modified 2D-gel compatible tris-sucrose buffer followed by a double TCA-acetone precipitation. This method omitted the use of toxic phenol which is widely used in the studies of plants proteins. Moreover, it shortens the lengthy extraction procedure of phenol extraction and back-extraction method and therefore reduced the extraction time (by 2h) while increased in protein yields (by 50%). Comparison of the 2D-gel images of the two extracts revealed that >60 extra protein spots were detected in the extract of our current method. The method was applied on the leaves of O. aristatus collected from six geographical areas in Malaysia. The correlation coefficient of each replicate gels from the six areas ranged from 0.70 to 0.90 indicating good reproducibility of the method. 相似文献
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Finnie C Steenholdt T Roda Noguera O Knudsen S Larsen J Brinch-Pedersen H Bach Holm P Olsen O Svensson B 《Phytochemistry》2004,65(11):1619-1627
The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome analysis was used to describe the water-soluble protein fraction of Golden Promise seeds in comparison with the modern malting cultivar Barke. Using 2D-gel electrophoresis to visualise several hundred proteins in the pH ranges 4-7 and 6-11, 16 protein spots were found to differ between the two cultivars. Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field and greenhouse-grown seeds were analysed. Four spots were observed to be increased in intensity in the proteome of greenhouse-grown seeds, three of which may be related to nitrogen availability during grain filling and total protein content of the seeds, since they also increased in field grown seeds supplied with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry. Phosphinothricin acetyltransferase was observed in three spots differing in pI suggesting that post-translational modification of the transgene product had occurred. 相似文献
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Aksu S Scheler C Focks N Leenders F Theuring F Salnikow J Jungblut PR 《Proteomics》2002,2(10):1452-1463
Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE. 相似文献
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Bryan Dunbar Thomas E. Elthon John C. Osterman Beth A. Whitaker S. Brian Wilson 《Plant Molecular Biology Reporter》1997,15(1):46-61
Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a
2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing,
and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing
from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag,
reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent
results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other
spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while
another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our
previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it
possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria. 相似文献
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Lorena Da Rós Gonçalves Márcia Regina Soares Fábio C.S. Nogueira Carlos Garcia Danielle Resende Camisasca Gilberto Domont Alfredo C.R. Feitosa Denise de Abreu Pereira Russolina B. Zingali Gilda Alves 《Journal of Proteomics》2010,73(7):1334-1341
Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis. 相似文献
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Schaefer H Chamrad DC Herrmann M Stuwe J Becker G Klose J Blueggel M Meyer HE Marcus K 《Biochimica et biophysica acta》2006,1764(12):1948-1962
In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation. 相似文献