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Development of a novel protein multi-blotting device

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Journal of Biomedical Science and Engineering 2010年第12期3卷 1125-1132页

作者： Amin M. Hagyousif Voon J. Chong Hiroki Yokota Stanley Y. P. Chien 不详

blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.

关键词： Western blotting Protein Transfer Multi-blotting PWM

Observation on Plant Leaf Transection by Gelatin blotting

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Journal of Northeast Agricultural University(English Edition) 2011年第3期18卷 7-11页

作者： TAN Dahai LI Fuheng WANG Xiaocen HUANG Fushan College of Life Sciences Northeast Agricultural University Harbin 150030 China Resources and Environmental Sciences College Northeast Agricultural University Harbin 150030 China

blotting was used to observe cell structures of leaf epidermis cells, and the key method of leaf transaction observation was paraffin section. The concentration, suitable solidification time, melting temperature of gelatin solution and the stain for the gelatin blotting were studied in this research. The results showed that the gelatin blotting could be used to study leaf transaction, it was benefit to make operation easily, and save time, money and so on

关键词： gelatin solution blotting leaf transaction observation

反向Northern blotting对小梅山猪下丘脑发育差异表达ESTs的鉴定

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江苏农业科学 2009年第3期37卷 22-24页

作者：周春宝潘孝青李霖丁家桐扬州大学动物科学与技术学院江苏扬州225009 江苏畜牧兽医职业技术学院江苏泰州225300 扬州教育学院江苏扬州225009

用mRNA差异显示技术从小梅山猪下丘脑不同发育阶段,分离并筛选出3个差异表达ESTs,分别命名为MS01、MS02、MS03。利用地高辛DNA标记与检测技术反向Northern斑点杂交,对分离出来的ESTs进行鉴定结果理想。此法避免了同位素放射性污染,操作... 详细信息

用mRNA差异显示技术从小梅山猪下丘脑不同发育阶段,分离并筛选出3个差异表达ESTs,分别命名为MS01、MS02、MS03。利用地高辛DNA标记与检测技术反向Northern斑点杂交,对分离出来的ESTs进行鉴定结果理想。此法避免了同位素放射性污染,操作简单,可防止DDRT-PCR过程中假阳性的出现,提高了试验准确性,是一种鉴定差异显示ESTs简单而有效的方法。

关键词：小梅山猪反向Northern blotting 下丘脑 ESTs 鉴定

疯牛病和羊痒病Western blotting检测方法的建立

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中国兽医学报 2007年第1期27卷 66-69,73页

作者：王辉暖赵德明宁章勇杨建民吴常德郝俊峰白玉王传武孟丽平中国农业大学国家动物海绵状脑病实验室北京100094

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索,并确定了最佳工作条件,结果表明:匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为... 详细信息

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索,并确定了最佳工作条件,结果表明:匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为恒压90V,分离胶电泳电压为恒压160V,转印的最佳电压和时间为恒压100V1.5h;封闭液为3%BSA时,封闭15min,封闭效果最好;AH6的最佳稀释度为1∶4000,4℃下孵育过夜,马抗鼠二抗的最佳稀释度1∶1000,室温下孵育30min。采用已确立的反应条件对样品进行检测并与Prionics-Check WEST-ERN进口试剂盒的检测结果比较,发现其敏感性为100%,特异性为99.4%,与进口试剂盒(100%,100%)无显著差异,这为国产试剂盒研制提供了条件。

关键词：疯牛病羊痒病 Western blotting

Prevalence of HIV Indeterminate Western Blot Tests and Follow-up of HIV Antibody Sero-Conversion in Southeastern China

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Virologica Sinica 2019年第4期34卷 358-366页

作者： Shouli Wu Min Gao Jian Zheng Pingping Yan Wei Wang Xiaoli Lu Yuefeng Qiu Yansheng Yan Fujian Provincial Center for Disease Control and Prevention Fuzhou 350001China Fujian Provincial Key Laboratory of Zoonosis Research Fuzhou 350001China School of Public Health Fujian Medical UniversityFuzhou 350004China Mengchao Hepatobiliary Hospital of Fujian Medical University Fuzhou 350025China Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention Wuxi 214064China Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology Wuxi 214064China Jiangsu Institute of Parasitic Diseases Wuxi 214064China

HIV-indeterminate Western blotting(WB)results are typically obtained in WB confirmatory assays,and the number of indeterminate samples may increase with the detection of HIV infections,which will present considerable challenges for the management of HIV/AIDS.Nucleic acid detection has been used as a laboratory test for screening suspected or indeterminate samples.However,the effectiveness of these assays for the differential diagnosis of HIV-indeterminate WB samples remained undetermined.In this study,210 subjects with HIV-indeterminate WB results were detected from 6360 positive HIV screening samples between 2015 and 2016 in southeastern China,in which HIV-indeterminate WB results accounted for 3.30%.The highest proportion of indeterminate results was observed in pregnant and lying-in women receiving physical examinations(16.67%),followed by that in voluntary blood donors(8.82%).The most common WB band patterns were p24,gpl60 and p24,and gpl60.The follow-up study revealed that the highest negative and positive conversion rates of HIV antibodies were in samples with a single p24 band(80.28%),and with gpl60 and p24 bands(86.21%),respectively.Among the Env,Gag,and Pol antibodies,samples with a Gag band showed the highest negative conversion rate(81.25%),whereas the highest positive conversion rate was observed in samples with an Env band(56.76%).In addition,quantitative and qualitative HIV nucleic acid testing exhibited the highest sensitivity(96.3%)and specificity(97.85%),respectively.Our results indicate a lower proportion of HIV indeterminate WB results in southeastern China compared to previous reports,and the follow-up re-examination of patients with HIV indeterminate results should be performed.Nucleic acid testing facilitates the identification of HIV infections.

关键词： Human immunodeficiency virus(HIV) HIV indeterminate result Western blotting Nucleic acid test Differential diagnosis

高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究

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首都医科大学学报 2016年第5期37卷 616-620页

作者：纪晓方李丽英常娜首都医科大学2011级基础医学专业北京100069 首都医科大学基础医学院细胞生物学系肝脏保护与再生调节北京市重点实验室北京100069

目的使用不同方法对蛋白质的核质分布进行检测并比较差异。方法细胞经免疫荧光染色后使用高内涵分析(high content analysis,HCA)对蛋白质的细胞质或细胞核总荧光强度进行分析;分别提取胞质及胞核蛋白,使用Western blotting法对核质组... 详细信息

目的使用不同方法对蛋白质的核质分布进行检测并比较差异。方法细胞经免疫荧光染色后使用高内涵分析(high content analysis,HCA)对蛋白质的细胞质或细胞核总荧光强度进行分析;分别提取胞质及胞核蛋白,使用Western blotting法对核质组分中的蛋白质含量进行鉴定。结果两种方法均可检测到蛋白质的出核转运;经定量分析,HCA法检测到的HuR蛋白质/核比升高倍数小于Western blotting法。结论使用HCA法检测蛋白质核质定位具有准确、客观、成本低、快捷、多参数等特点,不失为Western blotting的有效补充及替代方法。

关键词：高内涵分析 Western blotting 蛋白质核质分布

Treatment of chronic proliferative cholangitis with c-myc shRNA

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World Journal of Gastroenterology 2009年第1期15卷 95-101页

作者： Fu-Yu Li Nan-Sheng Cheng Jing-Qiu Cheng Hui Mao Li-Sheng Jiang Ning Li Sheng He Department of Hepatobiliary Surgery West China Hospital of Sichuan University Chengdu 610041 Sichuan Province China Key Laboratory of Transplantation Engineering and Immunology West China Hospital of Sichuan University Chengdu 610041 Sichuan Province China

AIM: To investigate the feasibility and effectiveness of c-myc shRNA in inhibiting the hyperplastic behavior and lithogenic potentiality of chronic proliferative cholangitis (CPC), in order to prevent stone recurrence and biliary restenosis.METHODS: An animal model of CPC was established by giving intralumenally 0.5 mL of c-myc shRNA. Then, the effects of c-myc shRNA on hyperplastic behavior and lithogenic potentiality of CPC were evaluated by histological observation, immunohistochemistry, real-time PCR and Western blotting for c-myc, proliferating cell nuclear antigen (PCNA), procollagen TM mucin5AC, enzymatic histochemistry for β-glucuronidase, and biochemistry for hydroxyproline in the diseased bile duct.RESULTS: Treatment with c-myc shRNA efficiently suppressed the hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expression of c-myc. More importantly, it decreased the lithogenic potentiality of CPC by inhibiting the expression of mucin 5AC and the secretion of endogenous β-glucuronidase. Further investigation indicated that c-myc shRNA-3 had a better inhibitory effect on CPC.CONCLUSION: Treatment with c-myc shRNA-3 can control CPC and reduce the lithogenic potentiality of CPC.

关键词： myc基因慢性增生性胆管炎治疗 blotting 葡萄糖醛酸酶增殖细胞核抗原结石复发

肝脏特异性表达载体的构建及Western blotting检测

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中国畜牧兽医 2011年第1期38卷 100-104页

作者：张利涛刘慧琳曹以诚华南理工大学生物科学与工程学院广东广州510006

研究靶向于肝细胞的组织特异性siRNA,实现siRNA基因治疗的组织特异性,可用于特异性治疗乙肝,突破RNA干扰技术在临床应用的一大障碍。用靶向于EGFP的siRNA(以下简称siEGFP)替代靶向于乙肝病毒保守区的siRNA,构建可以在肝细胞中特异性表... 详细信息

研究靶向于肝细胞的组织特异性siRNA,实现siRNA基因治疗的组织特异性,可用于特异性治疗乙肝,突破RNA干扰技术在临床应用的一大障碍。用靶向于EGFP的siRNA(以下简称siEGFP)替代靶向于乙肝病毒保守区的siRNA,构建可以在肝细胞中特异性表达的载体,用来表达siRNA。将目标载体分别转染肝癌细胞HepG2、乳腺癌细胞MDB-MB-231、人胚肾细胞293,在蛋白质水平上检验siRNA抑制的组织特异性。Western blotting结果证实,siEGFP在肝组织来源的细胞系HepG2中抑制效率明显高于其他两组细胞。构建的载体可以在肝癌细胞系中特异性的表达siRNA,而在其他组织细胞中不表达,实现了组织特异性。进一步将siEGFP替换为抑制HBV表达的siRNA,同样可以实现肝脏特异性的表达。

关键词： RNAi 组织特异性siRNA 肝脏特异性 EGFP Western blotting

Expression and location of α-fetoprotein during rat colon development

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World Journal of Gastroenterology 2009年第14期15卷 1738-1743页

作者： Xiao-Yan Liu Dan Dong Peng Sun Jun Du Luo Gu Ying-Bin Ge Department of Physiology Nanjing Medical University Nanjing 210029 Jiangsu Province China

AIM: To investigate the expression of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development. METHODS: Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reversetranscriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. RESULTS: The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested.demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

关键词：甲胎蛋白检测 SD大鼠 Western印迹甲胎蛋白mRNA 逆转录聚合酶链反应 blotting 免疫荧光染色发展中国家