共查询到19条相似文献，搜索用时 265 毫秒
1.
为了获得准确的奶牛冻精差异蛋白二维电泳(2-DE)分析结果,试验采用3种不同的方法,即改良TRIzol裂解法、改良三氯乙酸(TCA)-丙酮沉淀法和尿素盐酸胍裂解法,对奶牛冻精蛋白进行裂解,通过蛋白核酸浓度测定仪测定不同裂解法提取的蛋白量,用SDS-PAGE凝胶电泳检测不同方法提取的蛋白量。结果表明:随着精子数量的增加,裂解获得的蛋白量随之增加,改良TRIzol裂解法得到的蛋白量最高,尿素盐酸胍裂解法最低,改良TRIzol裂解法精子数为2.0×107、3.0×107和4.0×107个时蛋白量分别为(1.73±0.07),(1.94±0.05),(2.50±0.38)μg/μL,显著高于其他两种方法(P0.05);三种不同裂解方法上样量均为25μL,即含蛋白质26.21~62.5μg,不同上样蛋白量比较,精子数3.0×10~7个获得的条带最清晰、无粘连;蛋白条带分布的比较,改良TCA-丙酮沉淀法获得的条带多集中在50~85 ku区域,而改良TRIzol裂解法在30~50 ku区域和14~25 ku区域(精子数为3.0×10~7和4.0×10~7个)。说明精子数为3.0×10~7个时采用改良TRIzol裂解法能得到更丰富、更全面的精子蛋白质种类,有利于二维电泳(2-DE)分析寻找差异蛋白质,为后续试验奠定基础。 相似文献
2.
利用TCA-丙酮沉淀一裂解液溶解法提取朗德鹅肝脏蛋白质,然后采用常规双向电泳技术对提取的蛋白质进行分离,利用PDQuest软件分析电泳图谱,统计蛋白质点及比较凝胶重复性.试验得到(712±15)个蛋白质点,蛋白主要集中在pI 4.0～7.0和43.0～97.4 ku,重复胶的匹配点数为694个,蛋白质点匹配率为96％,蛋白量的相关系数为0.875.本研究建立了朗德鹅肝脏蛋白双向电泳技术,双向电泳图谱中蛋白位点的分辨率和重复性较高,为进一步研究其蛋白质组学奠定了基础. 相似文献
3.
细毛羊皮肤组织中毛囊蛋白质2-DE图谱的建立与初步分析 总被引：1，自引：1，他引：0
本研究以不同纤维直径的细毛羊皮肤组织中的毛囊作为试验材料,利用不同的提取方法、不同pH范围的IPG胶条以及不同的上样量,探索适用于细毛羊皮肤组织的双向电泳体系,建立细毛羊皮肤组织毛囊的2-DE凝胶图谱.凝胶图谱用ImageMaster 2D Platinum软件自动检测蛋白点比较不同纤维直径的细毛羊差异蛋白.结果显示,TCA/丙酮抽提法最适合细毛羊毛囊组织总蛋白质的提取.上样量为100 μL,选择18 cm、pH 4~7的线性IPG胶条,得到质量较好的双向凝胶电泳图谱,有35个蛋白差异点,为后续优质细毛羊的蛋白质组学研究工作奠定基础. 相似文献
4.
本研究以鸭瘟病毒感染鸭胚成纤维细胞为材料,围绕影响二维电泳因素进行全面探讨,以建立和优化鸭瘟病毒感染细胞蛋白质组二维电泳模型.结果表明,样品经过冷丙酮处理,水化液DTT浓度为30 mmol/L都有利于等电聚焦;采用PH5～8 IPG窄胶条和混合两性裁体电解质pH3～10/pH5～8为2/1比pH3～10 IPG宽胶条和单一两性载体电解质PH3～10分离蛋白时,各蛋白点间距较大,分辨率高,更有利于显示低丰度蛋白点;1.5 mg的蛋白上样量偏大,2～DE图像出现拖尾和水平条纹,部分相邻高丰度的蛋白重叠,且还掩盖了低丰度蛋白点.PDQuest7.40软件分析显示:17 cm PH5～8 IPG胶条电泳鸭瘟病毒感染细胞蛋白质组,银染可获得1 253个蛋白点,而考染却检测到388个蛋白点;重复试验仍获得清晰、稳定的2-DE图像,同一样本不同时期,考染可获得约348、331个蛋白点,蛋白点匹配率达88%,表明了鸭瘟病毒感染细胞蛋白质组二维电泳模型稳定、分辨率高、重复性好,为鸭瘟病毒蛋白组的进一步研究和新蛋白的发现提供了重要的研究方法. 相似文献
5.
雏鸡法氏囊蛋白质组学双向电泳技术的建立及其初步分析 总被引：2，自引：2，他引：0
为了建立并优化鸡法氏囊蛋白质组学的双向电泳技术体系,以不同日龄雏鸡的法氏囊组织为研究对象,用固相pH梯度胶条进行等电聚焦、SDS-PAGE垂直电泳,采用不同的样品制备方法,对上样量、水化、等电聚焦、胶条平衡和凝胶染色方法等进行一系列优化,并应用PDQuest8.0.1软件对图谱进行初步分析.结果显示法氏囊组织在pH 5～8范围、17 cm的2-DE胶上可以得到很好的分离,胶体考染后经PDQuest软件分析,在正常法氏囊组织可检测到800个以上蛋白点,不同2-DE图谱间蛋白点平均匹配率为83.5%,不同日龄雏鸡法氏囊存在有明显表达差异的蛋白质点37个,其中表达上调蛋白点17个,表达下调蛋白点11个,新增蛋白点5个,消失蛋白点4个,试验建立的鸡法氏囊组织蛋白质组双向电泳技术为法氏囊发育进化及其免疫功能的研究提供了新技术和方法. 相似文献
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精子功能相关精浆蛋白质的蛋白组学研究进展 总被引：1，自引：1，他引：0
精浆蛋白质对生殖过程中的精子功能有显著影响,包括顶体反应、精子获能、精子贮存、精子竞争和受精。精浆蛋白质的研究已有很长的历史,而且牛精浆蛋白、热休克蛋白、富含半胱氨酸分泌蛋白等几种主要的精浆蛋白质已被成功分离鉴定。蛋白质组学研究方法和技术的迅速发展,为全面解析精浆蛋白质组提供了新的研究策略,提高了研究者探索精浆蛋白质未知领域的效率。作者综述了精子功能相关精浆蛋白质研究的相关信息及近年来精浆蛋白质组的研究进展,从蛋白质水平上阐述了精浆蛋白质与精子获能、贮存及受精等功能的关系。 相似文献
7.
本研究以奶牛乳腺组织为对象,采用了普通离心、超速离心、2-D clean-up kit和TCA/丙酮沉淀方法制备蛋白质样品,经二维凝胶电泳(2-DE)获得蛋白表达图谱。根据凝胶图谱上蛋白质斑点判断图谱的质量,并运用PDQuest7.4软件分析图谱。结果显示:普通离心法制备的蛋白样品的凝胶图谱蛋白质斑点较为清晰,但横向条纹较多;超速离心法纯化蛋白样品的图谱,蛋白斑点清晰且可检测到较多的斑点;TCA/丙酮沉淀和2-D clean-up kit方法纯化蛋白样品的凝胶图谱条纹减少,蛋白斑点清晰,但可检测到的斑点数量有所减少。研究表明,超速离心方法纯化的蛋白质样品适合建立奶牛乳腺组织的蛋白质表达图谱。 相似文献
8.
为建立凡纳滨对虾血细胞蛋白质的双向电泳体系,实验将凡纳滨对虾血细胞蛋白质提取后,用双向电泳技术(2-DE)分离蛋白质,分别对蛋白质样品的制备方法、不同pH值范围IPG胶条、上样量等关键因素进行了探索和优化。结果显示,采用裂解液裂解-10%TCA/丙酮沉淀法制备蛋白质样品,使用17 cm pH值5~8的IPG胶条进行第一向等电聚焦电泳,第二向SDS-PAGE电泳采用浓度为12.5%的凝胶进行,上样量为每胶条200μg蛋白,第二向电泳后的凝胶采用硝酸银染色,扫描得到的凡纳滨对虾血细胞蛋白质双向电泳图谱蛋白质分离程度好、蛋白点清晰、分辨率高、横纹少等优点。文章建立并优化了凡纳滨对虾血细胞蛋白质组学的双向电泳技术体系,为进一步开展对虾等甲壳动物的蛋白质组学研究奠定了基础。研究表明,该双向电泳体系适用于凡纳滨对虾血细胞蛋白质的分离,可用于后续凡纳滨对虾血细胞蛋白质组学的研究。 相似文献
9.
旨在研究沼泽型水牛成熟前后卵泡内差异表达蛋白质的变化规律。采用双向凝胶电泳技术分离成熟卵泡液和未成熟卵泡液总蛋白质,建立和优化了卵泡液的双向电泳体系,并使用质谱鉴定差异表达蛋白点。结果显示,丙酮沉淀法处理得到的总蛋白质样品后,在24cm(pH 4～7)胶条且上样量350μg时得到分辨率较好的双向电泳图谱。软件分析得到11个差异蛋白点,以成熟卵泡液作为对照,5个蛋白点表达上调,3个蛋白点表达下调,1个蛋白点缺失,2个蛋白点在未成熟卵泡液中特异性表达。质谱成功鉴定出4个蛋白质:过氧化物酶-2、醛糖还原酶、牛纤维蛋白原的晶体结构、转甲状腺素蛋白。该研究建立了良好的卵泡液双向电泳体系,分析并鉴定一批水牛卵泡液差异蛋白质,对于研究水牛卵母细胞的发育微环境和成熟机制提供了新的研究线索。 相似文献
10.
建立稳定的山羊精子蛋白质双向电泳的技术平台，对精子上样量、蛋白质提取方法、二维电泳程序等相关技术都进行了优化，得到了相对清晰的冻精和鲜精的二维电泳图谱，并运用ImageMaster TM 2D Platinum软件进行了分析。结果表明：通过改进的热Trizol法提取山羊精子蛋白，当取精子3×10^7、上样量为150μL时能取得较好的电泳图谱，鲜精的蛋白位点重复性较高的有650个±10个，冻精的蛋白位点重复性较高的有600个±10个。冷冻导致精子蛋白丢失约50个，而且冻精多表现为大分子量蛋白的丢失，大部分分布于60～100kb之间，其中60～80kb之间蛋白丢失约18个，80～100kb之间蛋白丢失近20个，12～60kb之间蛋白丢失近12个。实验初步探明了山羊精子融冻前后精子的蛋白变化规律，为进一步了解精子冷冻损伤的确切机理奠定了基础。 相似文献
11.
草地藏系绵羊乳的组成及乳蛋白多态性 总被引：3，自引：0，他引：3
测定了63只草地藏系绵羊乳常规营养成分的含量,并用聚丙烯酰胺凝胶电泳分析了乳蛋白组分及乳蛋白多态性。结果表明:草地藏系绵羊脱脂乳中蛋白含量为(48.45±1.66)g/L,乳糖含量为(41.93±0.64)g/L,乳脂肪含量为(69.43±1.44)g/L;脱脂乳蛋白主要包括α-乳清蛋白、β-乳球蛋白、酪蛋白、免疫球蛋白等组分,酪蛋白的相对含量约为52%。乳中酪蛋白、β-乳球蛋白均未检测到多态性。试验检测到4种分子量类型的乳上皮粘蛋白(MUC1),分子量分别为214、209、207和205 ku。试验结果提示,草地藏系绵羊乳蛋白多态性较为贫乏。 相似文献
12.
This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10–25 kDa rank, the image analysis software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma. 相似文献
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Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting. 总被引：1，自引：0，他引：1
JA Cardozo M Fernández‐Juan JA Cebrián‐Pérez T Muiño‐Blanco 《Reproduction in domestic animals》2008,43(1):15-21
We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases. 相似文献
16.
CEA Souza AA Araújo JTA Oliveira AC Lima Souza JNM Neiva AA Moura 《Reproduction in domestic animals》2010,45(4):644-653
We have investigated the reproductive development of the tropically adapted Santa Inês ram, the most common hair sheep in Brazil. From 8 to 48 weeks of age, 16 animals were evaluated for body and testis growth, semen parameters, testosterone concentrations and seminal plasma proteins, using two‐dimensional SDS‐PAGE. Animals were weaned at 30 days and kept in feedlots thereafter, receiving hay, concentrate (18% of crude protein) and mineral supplement. Body weight increased from 12.3 ± 0.7 to 54.3 ± 1.6 kg between 8 and 48 weeks (p < 0.05), but changes in thoracic perimeter and scrotal circumference were non‐significant after 36 weeks (p > 0.05). The percentage of motile sperm increased slowly until 23 weeks and more rapidly after that age, but significant changes in progressive motility occurred after 25 weeks. Presence of abnormal sperm related inversely to age. Most significant changes in sperm concentration occurred between 38 and 44 weeks (0.38 ± 0.05 to 1.14 ± 0.24 × 109 cells/ml, p < 0.05) and testosterone reached its highest concentrations at 42 weeks, decreasing afterwards. Rams reached puberty at 28.2 ± 0.8 weeks. The number of protein spots on seminal plasma gels was similar from 15 to 18 weeks (45 and 47 spots; p > 0.05), increased until 24 weeks (141 spots) and 28 weeks (170 spots; p < 0.05) and remained without significant (p > 0.05) changes from 28 to 48 weeks (186 ± 10 spots). Furthermore, the intensity of selected spots on 2D maps increased (p < 0.05) between 15 and 28 weeks, which preceded or coincided with the main developmental changes in sperm motility and percentage of defective sperm in the ejaculates. These results will support future studies designed to characterize specific seminal plasma proteins whose expression relate to the development of testis, epididymis and accessory sex glands. 相似文献
17.
This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation. 相似文献
18.
A Shaliutina M Hulak P Li M Sulc B Dzyuba O Linhart 《Reproduction in domestic animals》2013,48(1):156-159
Seminal plasma of sterlet Acipenser ruthenus was evaluated using comparative proteomics to characterize its protein fractions and to determine any influence of multiple sperm collections on these proteins. An experimental group of fish was used, in which sperm was collected three times at 5 h intervals. Protein fractions of seminal plasma were determined by SDS‐gel electrophoresis (SDS‐PAGE) and two‐dimensional electrophoresis high‐resolution gels (2D). At all stripping times, five protein bands with molecular weights of 93, 53, 48, 33 and 28 kDa were identified using SDS‐PAGE. No significant differences (p > 0.05) in relative mass of protein bands among collections were observed. At the third collection, 20 protein spots were detected from the two‐dimensional gels, compared to 17 found at the first and second collections. Ten protein spots, from the third stripping, were analysed. Screening of these spots by mass spectrometric analysis showed positive results for spot 10. Direct comparison across public databases revealed sequence similarity with two hypothetical proteins, MCAG_00854 and IscW_ISCW011489. Differences in the seminal plasma protein fractions were found at the third stripping compared to the first two. It is hypothesized that these extra proteins after the third collection could be involved in some step of intracellular mechanism which is responsible for regulating of spermatozoa motility. However, protein identification revealed no significant distinction for any protein spot and protein sequences available in public databases. These results highlighted the need for a complete genome sequences for sturgeons. 相似文献