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Protein Extraction Methods for two-dimensional electrophoresis from Baphicacanthus cusia(Nees)Bremek Leaves-A Medicinal Plant with High Contents of Interfering Compounds

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Agricultural Sciences in China 2010年第10期9卷 1530-1537页

作者： XIANG Xiao-liang NING Shu-ju JIANG Xia GONG Xiao-gui ZHU Ren-lei WEI Dao-zhi School of Life Sciences Fujian Agriculture and Forestry University Fuzhou 350002 P.R. China School of Crop Sciences Fujian Agriculture and Forestry University Fuzhou 350002 P.R. China College of Chemistry and Materials Science Fujian Normal University Fuzhou 350002 P.R. China

Protein extraction is a critical step for two-dimensional electrophoresis （2-DE）. Different plant samples require different and adaptive protein extraction protocols. The leaves of medicinal plant, Baphicacanthus cusia （Nees） Bremek are notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds （especially the secondary metabolites and pigments）. This study was aimed to establish a routine procedure for the proteomic analysis ofB. cusia leaves, and a new protocol for the protein extraction was developed by optimizing trichloroacetic acid （TCA）/ acetone extraction method. The efficiency of this protocol was demonstrated by comparison with 3 published protein extraction methods （chloroform/acetone, Mg/NP-40, Tris-base/acetone）. The results showed that the optimized TCA/ acetone precipitation extraction method gave a relatively high protein yield （9.263 mg g^-1 fresh weight）, high-resolution separation, clear protein profiles, the highest proteins spots （1 31 t protein spots）, and displayed less contamination in 2- DE gels. Therefore, the results suggested that the optimized TCA/acetone method was the most effective among the 4 methods for B. cusia leaves.

关键词： Baphicacanthus cusia (Nees) Bremek medicinal plant protein extraction two-dimensional electrophoresis

two-dimensional electrophoresis Analysis of Proteins in Response to Cold Stress in Extremely Cold-resistant Winter Wheat Dongnongdongmai 1 Tillering Nodes

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Journal of Northeast Agricultural University(English Edition) 2012年第1期19卷 27-35页

作者： Cang Jing Yu Jing Liu Li-jie Yang Yang Cui Hong Hao Zai-bin Li Zhuo-fu College of Life Sciences Northeast Agricultural University Harbin 150030 China College of Communication and Electronic Engineering Qiqihar University Qiqihar 161006 Heilongjiang China College of Agriculture Northeast Agricultural University Harbin 150030 China

The overwintering survival ratio of the cultivar Dongnongdongmai 1 with strong cold-resistance in paramos of Heilongjiang Province in China are over 85%. The tillering nodes are the most important organs for overwintering survival of winter wheat, because there are more substances associated with cold resistance in tillering nodes than those in leaves and roots. Proteins in the tillering nodes of the cold-resistant cultivar Dongnongdongmai 1 grown under field conditions with or without any lowtemperature stress were analyzed by 2-dimensional electrophoresis and identified by mass spectrometry. In the range of pH 4-7, the expression of 37 proteins showed obvious difference （±more than two fold） in the proteomic maps of cold-stressed and non-stressed tillering nodes, including a new protein spot. All proteins exhibiting the difference in expression were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by a database search for protein identification and function prediction. Five groups of proteins were confirmed, namely stress-related proteins （22%）, metabolism-associated proteins （35%）, and signaling molecules （24%）, cell wall-binding proteins （5%）, unclear proteins （14%）. This indicated that tillering node cells supported the energy requirements of plant growth and stress resistance by signal transduction adapting to metabolism and structure.

关键词： Dongnongdongmai 1 proteome two-dimensional electrophoresis peptide mass fingerprinting

IDENTIFICATION OF DIFFERENTIAL PROTEINS IN UTERINE LEIOMYOMA BY two-dimensional electrophoresis

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Chinese Journal of Cancer Research 2006年第3期18卷 203-208页

作者：朱雪琼朱春丹呂杰强董克 Department of Gynecology The Second Affiliated Hospital Wenzhou Medical College Wenzhou 325000

Objective： To establish and optimize the two-demensional electrophoresis maps of uterine leiomyoma and to study the difference of global protein patterns between uterine leiomyoma and normal myometrium. Methods： Using two-dimensional electrophoresis followed by computer-assisted image analysis, the differential proteins between uterine leiomyoma and normal myometrium were compared. Results： The well-resolved and reproducible two-dimensional gel electrophoresis patterns of uterine leiomyoma and normal myometrium were established. Totally 1085±108 and 1103±151 protein spots were obtained by using the pH 4-7 IPG strips in uterine leiomyoma and normal myometrium map, respectively, of which 7 spots increased and 15 spots decreased in quantity in uterine leiomyoma compared with normal myometrium. Conclusion： The differentially expressed proteins are useful for studying the mechanism of the cause of uterine leiomyoma.

关键词： two-dimensional electrophoresis Uterine leiomyoma Proteome Differential expression

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

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Frontiers in Biology 2007年第3期2卷 309-313页

作者： LI Dongdong HE Shaoheng Department of Bioengineering Hainan UniversityHaikou 570228China Institute of Allergy and Inflammation Medical Collage of Shantou UniversityShantou 515031China

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current *** proteins were extracted by Trichloracetic acid(TCA)method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second *** spots of proteins were visualized by staining with Coomassie Brilliant *** analysis with software(ImageMaster 2D),122 different proteins were detected;isoelectric point(pI),Molecular weight(MW)and relative volume of each protein in the pollen were also *** is the first high-resolution,two-dimensional protein map of the pollen of Humulus scandens Lour in *** finding has built a solid foundation for identification,characteriza-tion,gene cloning and standardization of allergenic proteins in the pollen of Humulus scandens Lour for further studies.

关键词： Humulus scandens Lour pollen allergen proteins two-dimensional electrophoresis

Comparison of Three Methods of Protein Extraction from Dermatophagoides Pteronyssinus for two-dimensional electrophoresis

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Chinese Medical Sciences Journal 2009年第1期24卷 64-68页

作者： Jin-lu Sun Hong-yu Zhang Zhi-yi Guo Wan-tao Ying Xiao-hong Qian Jing-lan Wang Department of Allergy Peking Union Medical College Hospital Chinese Academy of Medical Sciences ＆ Peking Union Medical College Beijing 100730 China Experimental and Research Center Key Laboratory of Occupational Health and Safety for Coal Industry North China Coal Medical College Tangshan Hebei province 063000 China Beijing Institute of Radiation Medicine Beijing 100850 China

Obiective To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis （2-DE） analysis. Methods The extracts of Dermatophagoides pteronyssinus were prepared with Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid （BCA） assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. Results The concentrations of extracted protein by methods of Coca＇s solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight （LMW） range could be detected with the Coca＇s solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight （MMW） protein spots were absent. Several MMW protein spots （174-178 kD and 133 kD） and more LMW protein spots were detected withTrizol reagent method. Conclusions Among Coca＇s solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

关键词： house dust mite Dermatophagoides pteronyssinus allergen extraction two-dimensional electrophoresis

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by two-dimensional electrophoresis

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Acta Botanica Sinica 2002年第4期44卷 427-432页

作者：陈荣智翁清妹黄臻祝莉莉何光存武汉大学生命科学学院植物发育生物学教育部重点实验室武汉430072

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

关键词： rice recombinant inbred lines brown planthopper resistance two-dimensional electrophoresis

Establishment and Optimization of two-dimensional electrophoresis System for Spleen Proteome of Sillago sihama Forsskal

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Asian Agricultural Research 2019年第3期11卷 59-61页

作者： Junlin WANG Xiulan CHEN Zhonghong HU Jing LI Huanying PANG Yang HUANG Fisheries College Guangdong Ocean University，Zhanjiang 524088，China Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524088，China Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions，Zhanjiang 524088，China National Demonstration Center for Experimental Aquaculture Science Education，Zhanjiang 524088，China

Taking Sillago sihama Forssk?l as the research object,through the optimization of the extraction method,hydration and isoelectric focusing,a two-dimensional electrophoresis system was established for spleen proteome of S. sihama Forssk?l. The results showed that the twpstep hydration and isoelectric focusing method is better than the hydration and isoelectric focusing integrated method. The protein spots of the spleen of S. sihama Forssk?l were detected by two-dimensional electrophoresis mainly at pH 4. 0-7. 0. The establishment of this technical system will lay a foundation for further research on proteomics of S. sihama Forssk?l in the future.

关键词： Sillago sihama Forsskal Spleen Proteomics two-dimensional electrophoresis

Protein changes in human sperm demonstrated by two-dimensional electrophoresis before and after cryopreservation

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Asian Journal of Andrology 2003年第1期 68-68页

作者： Wen-Lei CAO, Zu-Qiong XIANG, Yi-Xin WANGShanghai Institute of Andrology, Shanghai Renji Hospital,Shanghai Second Medical University, Shanghai 200001, China

Objective: To establish the method of analyzing sperm protein by two-dimensional electrophoresis (2-DE) and compare the changes in protein expression before and after cryopreservation. Methods: Protein of 5×108 spermatozoa was solubilized in a 9.5 M urea lysis buffer. The sample was separated according to the isoelectric point (pI) by isoelectric focusing in the first dimension and according to the molecular weight (MW) by sodium dodecyl sulfate electrophoresis in the second dimension. The 2-D PAGE gels were stained with silver staining. Then these two protein profiles were compared. Results: The method of analyzing sperm protein by 2-DE was established and more than one protein was found to be changed after cryopreservation. Conclusion: More than one protein is changed after cryopreservation. 2-DE is an useful method to study sperm protein changes, but further improvement is needed for its practical application.

关键词： spermatozoa two-dimensional electrophoresis sperm preservation

Study on the Optimization of two-dimensional electrophoresis Technology System for Rapeseed Proteome

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Agricultural Science & Technology 2011年第5期12卷 625-629页

作者：王丽娟任学敏杜艳慧张其彬罗水忠姜绍通郑志合肥工业大学生物与食品工程学院安徽省农产品精深加工重点实验室安徽合肥230009

[Objective] The research aimed to establish the two-dimensional electrophoresis（2-DE）technology which was suitable for the rapeseed proteome research.[Method] Xiangyou 17 was as the *** sample preparation method,gel concentration and loading amount,*** 2-DE technology were optimized.[Result] The best extraction method of total protein of rapeseed was TCA-acetone method,and the protein spots on 2-DE map were the *** IPG strip（pH 3-10）and 12% gel were used,and the loading amount was 250 μg,the two-dimensional electrophoresis map with the clear background,good repeatability and high protein spot resolution was obtained.[Conclusion] The research laid the foundation for carrying out the rapeseed proteomics research.

关键词： Rapeseed Proteome two-dimensional electrophoresis System optimization