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.

A Rapid Electrophoresis Method on Agarose Gel to Characterise Dairy Protein Aggregates

Heat treatment of milk may cause whey proteins and caseins to form aggregates. These soluble and micellar aggregates and their other properties (size, composition, shape, etc.) can affect the techno-functionalities to the milk, conferring interesting or negative features depending on the application in dairy industries. In this study, we propose a new approach to characterise those protein aggregates. SDS-agarose electrophoresis is followed by the calculation of a retention factor (Rf) for each protein spot. Rf allows milk aggregates to be compared qualitatively under the same conditions. This method could be transposed to the dairy industry for a better knowledge of the milk subsequent to heat treatment.

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.

The protective effect of caffeine on DNA photosensitive damage:A gel electrophoresis

Agarose gel electrophoresis was performed to study interaction effect of caffeine on photosensitive injury of DNA caused by anthraquinone-2-sulphonic acid disodium(AQS),a model compound of strong photosensitizer,under 254 nm or 365nm UV irradiation Photosensitive injury of DNA induced by AQS under deoxidized condition was used as control.The results show that caffeine may resist effectively the injury effect of photosensitive damage and strong UV irradiation on DNA.The effects depend on the caffeine and AQS concentration,and irradiation time.Caffeine in concentration of 0.01-3.0μg/μL,may prevent DNA from damage induced by UV light,but caffeine in concentration of>5.0μg/μL accelerates the DNA damage.In particular,in the aqueous solution system of DNA,caffeine and AQS,at pH 6.25-7.35,the caffeine in concentration of 2.5-4.50μg/μL may resist the photosensitive injury of DNA caused by AQS under the deoxidized condition and exposure by 254 nm UV for 10 min.And caffeine in concentration of 5μg/μL would present a synergetic effect on the photosensitive injury of DNA.Possible molecular mechanism also is discussed.

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.

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.

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.

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.

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.

Sorting Semiconducting Single-Walled Carbon Nanotubes by Water-Soluble Polyfluorene Assisted Electrophoresis and Its Application in Field-effect Transistors

We report a considerably promising method based on agarose gel electrophoresis （AGE） to separate single-walled carbon nanotubes by adding a water-soluble polyfluorene （w-PFO） as surfactant into the agarose gel. In this effective method, the AGE/w-PFO gel network will trap more semiconducting single-walled carbon nanotubes （SWNTs） with the assistance ofw-PFO, for the strong interaction between w-PFO and semiconducting species. The optical absorbance, photoluminescence emission and resonant Raman scattering characterization were used to ver- ify the separation effect. The purity of separated semiconducting species is as high as （98±1）%. The demonstrated field effect transistors give the on/off ratio and mobility about 27000 and 10.2 cm^2·V^-1·s^-1, respectively.

Application of Two-Dimensional Electrophoresis in the Research of Retinal Proteins of Diabetic Rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.

Further Study on Separation of DNA Fragments by Capillary Electrophoresis by Quasi-interpenetrating Network of Polyacryamide and Polyvinylpyrrolidone with UV Detection

Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.

血浆脂蛋白琼脂糖凝胶电泳综合实验设计

血浆脂蛋白琼脂糖凝胶电泳实验是生物化学实验课程中的经典实验,但因其操作单一、等待时间过长,学生的收获感不强,有必要进行综合设计。把上课时间分为三个时段,在各个时段设计多种学习活动:串讲原理、做练习题使相关知识融会贯通;阅读文献,提出疑问;设置试误样品组、做相关实验,启发学生顿悟。通过这些学习活动,学生不仅学会了电泳技术的基本操作,还深刻理解了实验原理,强化了勇于探索的科学精神,体会了顿悟的快乐。

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.

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。

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。