一种点杂交膜预处理方法的建立及应用

旨在解决手工点样斑点杂交中样品点密度小、点样体积受限制和点样点不规整等问题,建立了一种新的点杂交膜预处理方法。首先根据预设点样点的大小、排列方式和密度,制作柱状凸起模具;其次将模具固定于杂交膜上,使模具凸起部位与膜紧密接触;再将熔化的石蜡涂布于膜上非模具接触部位,待石蜡凝固后移走模具,获得预处理杂交膜。以荧光染料IRDye800标记的抗体为样品进行直接点样检测,同时提取水稻、小麦和大豆的蛋白进行点杂交实验验证。结果显示,利用上述方法得到非点样部位具有疏水性的预处理NC膜和PVDF膜,其点样点位置、形状和大小固定,点样密度高。不同体积荧光染料标记抗体直接点样得到的荧光图像整齐规范,水稻、小麦和大豆蛋白与水稻看家蛋白抗体Anti-HSP杂交后均能检测到稳定信号。杂交膜经预处理后可最大限度实现点样点位置、大小和形状的统一,并能显著提高上样体积和点样密度。     

蛋白质的肽质谱指纹图分析方法的优化

肽质谱指纹图分析是一种常用的蛋白质的鉴定方法.为了提高这种方法鉴定蛋白质时序列覆盖率和准确度,以6个标准蛋白质为分析样品,对几种不同的酶解肽段的浓缩、脱盐和点样方法进行了检验和优化.结果发现,将酶解肽段的浓缩体积控制在5μl以下和采用10mmolL柠檬酸铵缓冲液板上脱盐能提高蛋白质鉴定的准确度;在点样的时候,采用先点样品再点基质的方法能明显提高匹配肽段的个数和信噪比.这些优化的样品制备方法明显地提高了MALDITOF质谱肽质谱指纹图分析方法鉴定蛋白质的可靠性.     

哺乳类基因工程的一些实验操作 实验4 琼脂糖凝胶电泳

一、琼脂箱凝胶的制备 1．电泳槽与点样孔梳的选择进行琼脂糖凝胶 电泳的电泳槽通常有水平板型和垂直板型两种。这里 我们选择较简便，应用最广的平板型电泳槽。点样孔 杭可根据样品数目、点样量等作出选择。对于一定量 的样品来说．，宽而薄的梳子可产生较细而平直的带，适 合于酶切图谱的分析等，较狭的梳子可增加待测样品 检出的灵敏度，适合于印迹杂交检测哺乳类基因组中 单拷贝基因或DNA样品的量较少等情况。点样孔梳 选定后，再根据凝胶的厚度，就可算出每一点样孔中可 以加入的样品体积。     

cDNA微阵列与寡核苷酸芯片的制备方法

cDNA微阵列和寡核苷酸芯片是常见的合成后点样的DNA微阵列。点样方法主要是通过物理吸附或共价结合的方式将探针固定于载体上,本总结了近年来国内外献报道的cDNA微阵列制备方法;在多聚赖氨酸包被的玻璃基片表面制备cDNA微阵列;用琼脂糖包被的玻璃基片制备cDNA微阵列;在氨基或醛基修饰的玻璃基片表面制备cDNA微阵列;寡核苷酸芯片的制备方法;氨基修饰的玻片与5′末端带氨基的寡核苷酸探针通过不同的linker连接;硅烷化寡核苷酸直接点样于玻片上制成寡核苷酸微阵列;硫代寡核苷酸通过二硫键与巯基修饰的玻片连接;水凝胶芯片固定寡核苷酸。丙烯酰胺硅烷化的基片与5′丙烯酰胺修饰的寡核苷酸连接。并展望了基因芯片的应用前景。     

梯度稀释及微量点样法在环境γ-变形菌分离中的应用

[目的]细菌分离中,梯度稀释及微量点样法可较好地保持细菌的原位丰度,理论上对优势菌的分离有较高的概率;应用梯度稀释及微量点样法分离γ-变形菌是一个有趣的研究点。[方法]应用梯度稀释及微量点样法,结合不同的分离条件,对7种环境样品进行γ-变形菌分离。[结果]从7种环境样品中成功分离52株细菌,经鉴定γ-变形菌为42株,占分离细菌总数的80.77%,呈现明显的γ-变形菌偏好性;已获得的γ-变形菌分属于8个不同的属,优势菌属为Klebsiella、Enterobacter、Escherichia,占已分离γ-变形菌的78.58%。[结论]实验所应用的梯度稀释及微量点样法对γ-变形菌的分离呈明显的偏好性(P0.05),是环境γ-变形菌分离方法的重要补充,该方法适用于环境优势菌的分离尝试。     

生物芯片高效点样微悬臂梁探针的微细加工

生物芯片技术是 90年代初发展起来的一门新兴技术 ,将给 2 1世纪生命科学和医学研究带来一场革命。其中生物芯片中的微阵列分析最为关键 ,尤其受到人们的关注和研究。微阵列形成主要由各种探针接触式点样而得的。微机电系统(MEMS)的兴起和发展为点样探针的制备提供了简单而有效的微细加工技术。介绍了基于MEMS技术的SiO₂ 基微悬臂梁探针的设计和制备 ,并用这种探针进行Cy3标记链亲和素点样。结果表明这种微悬臂梁探针可以点样 2～3μm的样点 ,并且一次取样可以点样至少 3000个点,从而实现高价生物微阵列点样。     

口蹄疫等5种动物病毒基因芯片检测技术的研究

用分子克隆方法获得口蹄疫病毒、水泡性口炎病毒、蓝舌病病毒、鹿流行性出血热病毒和赤羽病病毒各一段高度保守的基因片段,用芯片点样仪点样到包被过的玻璃片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测,可同时诊断上述5种动物传染病,此方法不但快速、准确、敏感,而且可同时进行多种病毒的检测,达到大批动物高通量检疫的目的。     

DNA微阵列基片的化学处理

对于合成后点样的DNA微阵列 ,基片的表面化学处理非常重要。它直接影响到样品与基片的结合效率 ,进而影响杂交结果。基片表面的各种化学修饰方法多种多样 ,物理吸附主要以赖氨酸包被为主。共价结合通常使用同源偶联分子或异源偶联分子 ,还可以在基片表面组装线状、分支状连接分子或包被琼脂糖。着重介绍了DNA微阵列的制备 ,即样品如何固定到玻璃基片上。总结了不同类型基片表面的化学修饰方法以及DNA与基片的化学结合。     

硅胶薄层层析技术在分离纯化鱼类性类固醇激素中的应用

应用硅胶薄层层析技术结合放射照相定位方法,进行鱼类血清和性腺离体培养液中六种性类固醇激素的分离、纯化。文章具体介绍了样品抽提,点样,层析溶剂系统选择,照相定位,洗脱等技术步骤。     

为什么点样孔会发亮

分析和检测核酸的一般办法是琼脂糖凝胶电泳．并且用溴化乙锭(EB)显色。除目的条带以外,人们还经常发现点样孔上或点样孔四周也能被EB染色而发亮。许多学生就此问过老师。老师们一般将其解释为核酸样品不纯,有蛋白质污染或有其他杂质污染。还有些人认为这是琼脂糖凝胶本身的因素造成的。但真正的原因是什么,     

Protein modification: docking sites for kinases.

The substrate specificities of protein kinases have been found, in many cases, to be determined at least in part by short regions within the substrate known as docking sites. Docking sites are specific and modular, and can dramatically increase the efficiency of phosphorylation.     

Computational modelling and analysis of mechanical conditions on cell locomotion and cell–cell interaction

Between other parameters, cell migration is partially guided by the mechanical properties of its substrate. Although many experimental works have been developed to understand the effect of substrate mechanical properties on cell migration, accurate 3D cell locomotion models have not been presented yet. In this paper, we present a novel 3D model for cells migration. In the presented model, we assume that a cell follows two main processes: in the first process, it senses its interface with the substrate to determine the migration direction and in the second process, it exerts subsequent forces to move. In the presented model, cell traction forces are considered to depend on cell internal deformation during the sensing step. A random protrusion force is also considered which may change cell migration direction and/or speed. The presented model was applied for many cases of migration of the cells. The obtained results show high agreement with the available experimental and numerical data.     

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.     

Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.     

Medicinal mushrooms: a rapidly developing area of biotechnology for cancer therapy and other bioactivities

Historically, medicinal mushrooms (basidiomycetes) have been shown to have profound health promoting benefits and recent studies, which are reviewed here, are now confirming their medical efficacy and identifying many of the bioactive molecules. Methods of large-scale cultivation by solid substrate and liquid culture fermentations are also briefly described.     

Risks associated with environmental enrichment: intestinal obstruction caused by foraging substrate

Questions are occasionally asked about the safety of enrichment techniques, considering that many novel ways are frequently employed to ensure environmental complexity. A juvenile male vervet monkey was found with a phytobezoar of straw obstructing the sigmoid colon. The straw was foraging substrate, which is used in communal cages. Due to the extent of the resulting necrosis in the sigmoid and descending colon, the monkey had to be killed. This is the only individual to have suffered a harmful effect from the foraging substrate from amongst 120 vervet monkeys, which have been permanently housed on straw for over 5 years.     

Probing plant polyketide biosynthesis.

The first structure of a chalcone synthase has been determined. Enzymes in this family have broad substrate specificities and participate in the biosynthesis of many secondary plant products, including compounds that provide defense against pathogens and precursors to the bitter acids in hops that flavor beer.     

Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs

Protein arginine methyltransferases (PRMTs) are enzymes that are involved in many biological processes. Several studies have shown that the identity of the N-terminal fusion tag affects the substrate selectivity of PRMTs. Therefore, to accurately study substrate recognition, it is imperative that a tagless PRMT be used. However, cleavage of tagged PRMTs has been problematic. We have developed a successful method by which untagged PRMTs can be made using a tobacco etch virus (TEV) cleavage site at the N-terminal domain. This method may be useful for cleaving other challenging target proteins that have the TEV protease recognition site.     

F-box蛋白家族的功能研究进展

F-box蛋白是一类含有F-box基序(motif),在泛素介导的蛋白质水解过程中具有底物识别特性的蛋白质家族.这类蛋白质在细胞时相转换、信号传导、发育等多种生理过程中都具有重要功能.     

N- and C-terminal degradomics: new approaches to reveal biological roles for plant proteases from substrate identification

Proteolysis is an irreversible post-translational modification that regulates many intra- and intercellular processes, including essential go/no-go decisions during cell proliferation, development and cell death. Hundreds of protease-coding genes have been identified in plants, but few have been linked to specific substrates. Conversely, proteolytic processes are frequently observed in plant biology but rarely have they been ascribed to specific proteases. In mammalian systems, unbiased system-wide proteomics analyses of protease activities have recently been tremendously successful in the identification of protease substrate repertoires, also known as substrate degradomes. Knowledge of the substrate degradome is key to understand the role of proteases in vivo. Quantitative shotgun proteomic studies have been successful in identifying protease substrates, but while simple to perform they are biased toward abundant proteins and do not reveal precise cleavage sites. Current degradomics techniques overcome these limitations by focusing on the information-rich amino- and carboxy-terminal peptides of the original mature proteins and the protease-generated neo-termini. Targeted quantitative analysis of protein termini identifies precise cleavage sites in protease substrates with exquisite sensitivity and dynamic range in in vitro and in vivo systems. This review provides an overview of state-of-the-art methods for enrichment of protein terminal peptides, and their application to protease research. These emerging degradomics techniques promise to clarify the elusive biological roles of proteases and proteolysis in plants.