Optimization of SSR-PCR Non-denatured Polyacrylamide Gel Electrophoresis Conditions in Kernelled Apricot

[Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were selected as experimental materials to screen the repeatable SSR loci with high polymorphism by the use of SSR markers combined with non-denatured polyacrylamide gel electrophoresis.And the effect of different factors on electrophoresis conditions was compared to explore the optimal SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Result]The optimal non-denatured polyacrylamide gel electrophoresis conditions for SSR-PCR were established as follows：polyacrylamide gel concentration 6%,the ratio of acrylamide to bisacrylamide 29∶1,electrophoresis at 1 000 V for 2-3 h,and staining for 15 min within 0.1% AgNO3.[Conclusion]The optimum electrophoresis system has provided some technical foundations to further study the phylogenetic relationship of kernelled apricots by SSR markers.

Separation of non-denatured proteins using semi-crosslinked polyacrylamide capillary gel electrophoresis

This work presents an approach to build a high-performance, low-viscous and replaceable separation matrix, semi-crosslinked polyacrylamide （semi-CPA） capillary gel electrophoresis. Non- denatured basic proteins, such as lysozyme, cytochrome C, ribonuclease A and trypsin were separa- ted. The impacts of monomer and cross-linker concentrations on protein separation were studied, and the ability of dynamic capillary inner wall coating was demonstrated. The UV absorption interfer- ence by semi-CPA gel matrix was successfully overcome by a partial filling technique, which results in sensitivity 20 times higher than other protein separation method. The excellent separation ability, reproducibility and dynamic coating ability made semi-CPA an ideal separation media in both capillar- y electrophoresis and microfluidic chip separation scheme.

Quantitative Detection of Inositol Hexakisphosphate (InsP6) in Crop Plants Using Polyacrylamide Gel Electrophoresis (PAGE)

Inositol phosphates are essential for cell development and signaling in all living organisms. Inositol hexakisphosphate (InsP6) is the most abundant phosphoinositol in both plants and animals. While the concentration of inorganic phosphorous (Pi) is often limited in soil, some plants overcome this limitation by creating a phosphate reservoir that serves as a source of Pi during phosphate deficiency. Although this strategy benefits plant development and signaling under adverse environmental conditions, excessive accumulation of Pi in crop plants has raised serious concerns about its toxicity and ill effects on human health. Consumption of crop plants with high InsP6 content or food products made from these crops is found to reduce nutrient intake significantly by way of chelating essential metal cations in human and livestock fed by such plants. Therefore, it is necessary to determine InsP6 contents in crop plants. Several methods have been developed for the screening and detection of InsP6 in plants. These detection methods however, are complex, labor-intensive, and often provide inaccurate results. We have developed a fast, reliable, and cost-effective method for the detection and quantification of InsP6 in plants using polyacrylamide gel electrophoresis (PAGE) with potential applications in industry, quality control labs, and research projects.

Research on Sex and Tissue Differences in Isozymic Phenotype of Varicorhinus macrolepis through Isozyme Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male and female Varicorhinus macrolepis. It had been found for the first time that the phenotype of malate dehydrogenase (MDH),acid phosphatase (ACP) and superoxide dismutase (SOD) showed difference between male and female V. macrolepis,and there was no difference among different individuals in the same sex. Therefore,the electrophoresis band of malate dehydrogenase,acid phosphatase and superoxide dismutase could be used as an indicator for the identification of gender and tissues of V. macrolepis,which would provide basic data for the developmental genetics,variety improvement and directed breeding of V. macrolepis groups,thus facilitating the development and protection of this valuable fish species.

Characterization of Agro-diversity by Seed Storage Protein Electrophoresis:Focus on Rice Germplasm from Uttarakhand Himalaya,India

The characteristics of 48 rice varieties from Uttarakhand Himalaya, India were detected by morphological and biochemical markers. The grains of the selected rice varieties varied in their morphological （grain length, grain width and grain weight） and biochemical characters （sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）. Based on the presence of 70, 65, 60, 57, 37-39, 22-23, 13 and 10 kDa protein bands in the 48 rice varieties, seven types of profiles were identified. An unweighted pair group average method with arithmetic mean （UPGMA） dendrogram based on cluster analysis of genetic similarity of the protein bands showed two distinct groups with 1%-78% similarity coefficients. The presence of characteristic bands in selected varieties is a useful parameter for identification of rice germplasm.

Isolation and purification of guinea pig inner ear antigens by preparative polyacrylamide gel electrophoresis

Objective To isolate and purify the subcomponents of guinea pig inner ear antigens for further study on the autoimmunity of the inner ear.Methods Inner ear tissues were homogenized in phosphate buffered saline containing 0.1% SDS, and then frozen and defrosted repeatedly to extract inner ear antigens. Preparative polyacrylamide gel electrophoresis was used to separate the subcomponents of inner ear antigens. Following electrophoresis,the protein bands were localized by rapid staining and destaining.Results The major protein bands were clearly distinct when 3 mg of crude inner ear antigens was loaded,and the three major subcomponents (31, 42- 45 and 60 kD proteins) accounted for about 25.99%,21.91% and 21.10%, respectively.Conclusion Preparative polyacrylamide gel electrophoresis can be used to purify the major subcomponents of inner ear antigens.

东方田鼠雌雄个体不同器官组织中苹果酸脱氢酶比较分析

为了解东方田鼠(Microtus fortis)雌雄个体不同器官组织中苹果酸脱氢酶(malate dehydrog enase,MDH)的活性及分布,采用非变性不连续的聚丙烯酰胺凝胶电泳对东方田鼠雌雄个体的心脏、肝脏、肾脏、肌肉、脑、肺脏中的革果酸脱氢酶进行分离,建立电泳图谱并进行比较分析.结果表明,东方田鼠雌雄个体中革果酸脱氢酶分离出迁移率为0.019~0.514的23条谱带,苹果酸脱氢酶在东方田鼠雌雄个体的6种器官组织中均有表达,东方田鼠雌雄个体之间及同一个体不同器官组织之间酶的活性及分布有差异.本研究为东方田鼠实验动物化及农林鼠害的防治提供了基础的生化数据资料.

A green method of staining DNA in polyacrylamide gel electrophoresis based on fluorescent copper nanoclusters synthesized in situ

The safety of nucleic acid staining dyes has long been recognized to be a problem. Extensive efforts have been made to search for alternatives to the most popular but toxic staining dye, ethidium bromide （EtBr）. However, so far no staining method that can be guaranteed to be suffidently safe has been developed. In this paper, we report a green staining method of DNA in polyacrylamide gel electrophoresis, where in situ synthesis of DNA-templated fluorescent copper nanoclusters （CuNCs） in the gel is achieved to make the DNA bands visible under UV light. Moreover, a comprehensive study of the performance of this staining method has been conducted and the experimental results show that it has favorable sensitivity, stability, and usability. Meanwhile, in our animal experiments, the two reagents （copper sulfate and ascorbic acid） as well as the synthesized CuNCs have been proven to be non-toxic in contact with skin. In addition, all the reagents employed in this work are readily available and low cost, and the procedure is simple to carry out. Therefore, this novel staining method based on the in situ synthesis DNA-templated fluorescent CuNCs has many potential applications.

乳及乳制品中乳铁蛋白含量的快速SDS-PAGE荧光检测方法

该研究采用新型的Chromeo P503(P503)染料替代考马斯亮蓝染色方法,以克服脱色染色步骤繁琐、耗时长(≥5 h)的不足,建立一种乳及乳制品中乳铁蛋白的快速简便十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis,SDS-PAGE)检测方法。P503染料标记蛋白产生强烈的红色荧光发射,在30 min内一步操作即可完成,并且进行脱铁处理后消除乳铁蛋白铁饱和度对于荧光信号的干扰,保证定量的准确性。根据凝胶成像图中78 ku蛋白条带是否存在及其灰度值的大小,实现乳铁蛋白的定性与定量检测。在优化的实验条件下,线性范围为15~75μg/mL,检出限为5.11μg/mL,加标回收率在75.79%~108.09%之间,表明该方法具有较高的灵敏度和准确性。SDS-PAGE和P503结合无需进行肝素亲和柱前处理,便可直接应用于乳及乳制品中乳铁蛋白快速简便的准确定量检测,P503染料标记还可以用于其它乳蛋白的SDS-PAGE快速低成本定量检测,从而为乳品质量控制和营养评价及加工工艺研究提供有力工具。

六堡茶茶褐素中酚类化合物及蛋白质组分分析

本研究通过甲醇提取茶褐素,将其分为可提取部分(记为FTB)和不可提取部分(记为BTB),采用傅里叶变换红外光谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白质印迹和超高效液相色谱-高分辨质谱方法开展组分分析。结果表明,茶褐素为多羟基的芳香族物质,含有蛋白质、氨基酸和肽等含氮化合物;在蛋白质分子质量63~75 kDa附近具有条带,总体为弥散型的长条且可能被多酚等物质修饰;非靶向代谢组学共鉴定出297种化合物,以差异倍数(fold change,FC)>2或FC<0.5和P<0.05为标准进行筛选,发现BTB与FTB之间有137种化合物存在显著差异,主要为酚类化合物(37种);通过主成分分析和正交偏最小二乘判别分析获得了FTB与BTB的5种酚类化合物(儿茶素、2,3-二羟基苯甲酸、没食子酸、4-羟基香豆素和咖啡酸),它们不仅可以区分FTB和BTB,还有可能是茶褐素的主要不可提取酚类。本研究解析了六堡茶茶褐素的多酚谱及其特征性多酚,提出了蛋白质-多酚复合物的推论,有助于丰富黑茶茶褐素中蛋白质的相关研究,推动黑茶茶褐素的物质组成及功能活性的进一步研究。

Conformer Analysis of a Biological G-Quadruplex Element in the Human c-MYC Promoter by Native Polyacrylamide Gel Electrophoresis

The G-quadruplexes undergo complex folding and conformation exchanges.G-quadruplex stability is substantially influenced by sequence,bufer and temperature.Mutational analysis together with nuclear magnetic resonance spectroscopy(NMR),X-ray crystallography and circular dichroism spectroscopy has been proved to be a powerful approach for G-quadruplex structural analysis.Herein,we used DNA sequence mutation and native polyacrylamide gel electrophoresis to investigate the topology and conformations of a G-quadruplex model molecule Pu18 found in the human c-MYC promoter.The guanines(G6,G9 or G18)which were not contributable to G-tetrad formation in c-MYC Pu18 sequence were mutated to thymine or adenine.We screened the buffer and temperature of gel electrophoresis for Pu18.Gel electrophoresis showed that two of the four conformers of c-MYC Pu18 in 100 mM K+buffer were resolved,which was in accordance with the conformations as determined by the 1 H NMR spectra in previous studies.This technique is expected as a general methodology for its easy operation and low cost to facilitate uncovering more yet unidentified G-quadruplex folds and functions,with the assistance of other analytical methods like NMR,X-ray crystallography and circular dichroism spectroscopy.

低压静电场处理对鱿鱼保鲜效果的影响

为探究低压静电场(low voltage electrostatic field,LVEF)处理对鱿鱼贮藏品质的影响,采用pH值、蒸煮损失率、挥发性盐基氮(total volatile basicnitrogen,TVB⁃N)和硫代巴比妥酸(thiobarbituric acid,TBA)等评价指标,以无电场处理的样品为对照组,研究比较低压静电场贮藏条件下的鱿鱼品质变化情况。结果表明,低压静电场处理能够改善鱿鱼的贮藏品质。随着贮藏时间的延长,LVEF组的样品pH值增加缓慢;贮藏期间对照组的蒸煮损失率、TVB⁃N值始终低于LVEF组,且当贮藏至15 d时LVEF组的TBA值远低于对照组的1.24 mg/kg;低压静电场对微生物繁殖有明显的抑制作用,贮藏15 d内LVEF组菌落总数均低于6 lg(CFU/g);在硬度、弹性、感官评价和蒸煮损失率上LVEF组均优于对照组。此外,十二烷基硫酸钠⁃聚丙烯酰胺凝胶电泳分析显示LVEF组条带未发生明显变化,故低压静电场处理能更好地保持鱿鱼品质。

6种铁线莲种质SSR遗传多样性分析

通过对河南25个居群的6种铁线莲种质进行遗传多样性分析,研究铁线莲种质资源分布和种群间的亲缘关系.筛选出6对引物,利用SSR标记技术对120个铁线莲样品进行遗传多样性分析.结果表明,25个居群的120个铁线莲的PIC值范围为0.619~0.738,有效等位基因数(Ne)为1.577±0.233,遗传相似系数在0.58到1.03之间,6对引物扩增出33条带,平均每个引物5.5条.同一种质的铁线莲优先聚为一类,同一种质亲缘关系更近,地理位置较近的亲缘关系也较近,25个居群的铁线莲具有丰富的遗传多样性.研究结果可为后续铁线莲种质创新和新品种选育提供参考.

Non-decoloured In-gel Digestion of Coomassie Blue-stained Proteins

A simplified method is presented for tryptic digestion of Coomassie brilliant blue(CBB)-stained proteins in polyacrylamide gels. Compared with conventional methods, the proposed method does not require a removal of the dye before digestion, and is thus faster and saves a lot of labor. The resulted digest can be analyzed by either RPLC/ESIMS or MALDI MS for identification of the protein in a conventional way. Model studies with bovine serum albumin(BSA) showed that 50 ng of the protein could be routinely identified. The simplified procedure displays a tendency to produce more incompletely cleaved peptides, which is favorable for improving the sequence coverage.

模拟酶解优化鸽血红蛋白抗氧化肽酶法制备

以鸽血红蛋白为原材料,采用在线数据库ExPASy Peptide Cutter中的蛋白酶模拟酶解,通过生物信息学工具筛选多肽潜在的生物活性、溶解性、毒性和致敏性,由此预测最适宜制备鸽血红蛋白源抗氧化肽的蛋白酶。设置不同的酶解时间、酶添加量和酶解温度作为单因素,探究不同酶解条件对酶解产物水解度和DPPH自由基清除率的影响。在单因素试验的基础上,通过响应面法优化试验,探究酶解法制备鸽血红蛋白抗氧化肽的最优工艺条件。结果表明,蛋白酶K为虚拟筛选出的最佳蛋白酶,最佳的制备鸽血红蛋白抗氧化肽的工艺为鸽血红蛋白浓度1 g/100 mL、pH8、酶解时间3 h、酶添加量415.54 U/g、酶解温度65℃。在此条件下,通过验证试验得出响应值DPPH自由基清除率为(33.21±0.68)%,与预测值相差较小,故而此条件为最优工艺。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳试验,发现最优工艺条件下鸽血红蛋白酶解后目的条带消失,进一步验证该方法为酶解鸽血红蛋白源抗氧化肽的最优工艺。

牛血抗菌肽的制备及应用

以新鲜黄牛血为原料,利用离心、裂解等方法提取抗菌肽,在单因素试验的基础上通过正交试验研究牛血离心转速、NH4Cl溶液与样品体积比、超声波功率和超声时间对粗抗菌肽得率的影响。结果表明:最佳提取条件为牛血离心转速3 500 r/min、NH4Cl溶液与样品体积比3∶1、超声功率600 W、超声时间8 min,验证得粗抗菌肽得率可达0.008 5%;进一步采用聚丙烯酰胺凝胶电泳法确定抗菌肽的分子量,并通过电洗脱分离纯化的抗菌肽,结果表明抗菌肽的分子量在14 kDa左右,电洗脱回收率为82%。

High-throughput Three-dimensional Gel Electrophoresis for Versatile Utilities: A Stacked Slice-gel System for Separation and Reactions (4SR)

A novel high-throughput system, called the stacked slice-gel system for separation and reactions （4SR）, was developed for the analysis of DNA/RNA and protein/peptide. The system provides a novel three-dimensional gel electrophoresis approach that exploits the property of stacked slice gels. It allows multiple samples simultaneously to react as well as to be separated, offering a two-dimensional （m×n） sample loading system. For this purpose, high-throughput multi-micro vessels （MMVs） containing variable numbers of wells （100 wells in this paper） have been used, which are made of 25 mm square-size polyacrylamide gels. Furthermore, after electrophoretic separation, a slice gel containing a desired sample can be easily removed and proceeded to the next step. Different biological reactions as well as successive separation of products were effectively carried out dealing with DNA/RNA and protein/peptide. It shows that this system has a diversity of potentials to be developed.

Optimization of Extraction Methods of ROP GTPase from Young Leaves of Wheat

All of the Rho GTPase seems to form a special sub-family,because such a sub-family has so far only found in plants,and then named Rop GTPase,which directly involved in and regulated of muscle actin cytoskeletal reorganization,such as a series of signal transductions.The efficient purification technology and the means of ROP GTPase in wheat are the key basis of the studies on its functions and prosperities.And it has a very important theoretical and practical significance in the signal transduction and F-act...

鉴定水稻品种的微卫星标记筛选

选用58个水稻微卫星标记,对目前应用较广的28份杂交水稻亲本材料进行多态性分析,并比较了其中14个标记应用3.0%琼脂糖凝胶电泳和6.0%非变性聚丙烯酰胺凝胶电泳的检测结果。研究表明,所应用的标记可将所有供试水稻材料区分开,不同标记的多态性检测能力差异较大;两种检测方法所得结果高度一致,但非变性聚丙烯酰胺凝胶电泳分辨率较高、带型清晰,能更准确反映水稻材料间的差异。对于一般水稻材料而言,选用12个标记可将不同材料区别开的几率大于99.9%,但要区分遗传相似性较高的材料,则可能需挑选高多态性标记和(或)增加检测的标记数。

聚丙烯酰胺凝胶快速、高效银染方法的建立

聚丙烯酰胺凝胶电泳目前已经广泛应用在SSR标记、SNP标记以及遗传图谱的构建等过程中,但是一直以来凝胶银染方法由于染色时间长、染色步骤繁琐,使得对于开展大批量的实验研究极为不利。文章通过对常规方法的改良,建立了一个银染步骤只需要10min,整个染胶过程只需约20min的快速、高效的银染方法。