目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有...目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。展开更多
[目的]探讨麦洼牦牛转铁蛋白(Transferrin,TF)的结构及生理功能。[方法]采用RT-PCR方法,从麦洼牦牛心脏克隆出TF基因,并运用多种生物信息学软件对其进行核苷酸序列及蛋白质结构分析。[结果]克隆得到的麦洼牦牛TF基因开放阅读框为780 bp...[目的]探讨麦洼牦牛转铁蛋白(Transferrin,TF)的结构及生理功能。[方法]采用RT-PCR方法,从麦洼牦牛心脏克隆出TF基因,并运用多种生物信息学软件对其进行核苷酸序列及蛋白质结构分析。[结果]克隆得到的麦洼牦牛TF基因开放阅读框为780 bp,编码259个氨基酸,分子质量为28.33 k Da,理论等电点8.57,不含跨膜区域,存在信号肽序列,二级结构以随机卷曲和α-螺旋为主,无β-折叠,同源建模预测出牦牛TF蛋白与猪血清转铁蛋白有相似的三级结构,同源性可达71.91%。[结论]成功克隆了麦洼牦牛TF基因并分析了其蛋白质结构,为进一步研究TF蛋白功能提供理论基础。展开更多
为了更好地了解市售椰浆中椰子蛋白质量状况,本研究以市售的10种椰浆为样品,采用反向高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium laurylsulfonate-polya...为了更好地了解市售椰浆中椰子蛋白质量状况,本研究以市售的10种椰浆为样品,采用反向高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium laurylsulfonate-polyacrylamide gel electrophoresis,SDS-PAGE)对提取蛋白进行分析,采用基质辅助激光解吸电离飞行时间串联质谱(matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry,MALDI-TOF/TOF-MS)对蛋白进行鉴定。结果表明:椰浆RP-HPLC约在10.4 min和11.9 min分别出现1个色谱峰,大多数样品峰面积大小都与蛋白标签含量一致,SDS-PAGE条带数量和颜色的深浅与RP-HPLC得到的峰面积结果一致,个别样品标签含量高,SDS-PAGE能分离出的蛋白条带数量也较多,但RP-HPLC的椰子蛋白峰面积小,经MALDI-TOF/TOF-MS鉴定,部分条带没有鉴定出椰子有关的肽段或蛋白。研究结果为椰浆蛋白的食品安全与质量控制提供了方法参考和示范。展开更多
With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and bi...With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and biofunctionalized nanoparticles, a series of novel principles, methods and techniques have been exploited for bioanalytical and biomedical studies. This review mainly discusses the applications of molecular beacon probes and biofunctionalized nanoparticles-based technologies for realtime, in-situ, highly sensitive and highly selective protein analysis, including the nonspecific or specific protein detection and separation, protein/DNA interaction studies, cell surface protein recognition, and antigen-antibody binding process-based bacteria assays. The introduction of molecular beacon probes and biofunctionalized nanoparticles into the protein analysis area would necessarily advance the proteomics research.展开更多
With the sequencing of human genome almost complete, human genome project enters the post-genome-sequencing era. Compared to genomics, the analysis of proteome is rather difficult since in human cells there are around...With the sequencing of human genome almost complete, human genome project enters the post-genome-sequencing era. Compared to genomics, the analysis of proteome is rather difficult since in human cells there are around 200000 proteins, which are expressed at any time at different levels of concentration with a wide dynamic range. In addition, a given proteome is not static but dynamic, being defined by a combination of the cell's genome, environment, and even its history. To meet the higher and higher requirements from proteome analysis, in our recent work, we have developed some capillary electrophoresis (CE)-based techniques for the analysis of complex protein samples.展开更多
文摘目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。
文摘[目的]探讨麦洼牦牛转铁蛋白(Transferrin,TF)的结构及生理功能。[方法]采用RT-PCR方法,从麦洼牦牛心脏克隆出TF基因,并运用多种生物信息学软件对其进行核苷酸序列及蛋白质结构分析。[结果]克隆得到的麦洼牦牛TF基因开放阅读框为780 bp,编码259个氨基酸,分子质量为28.33 k Da,理论等电点8.57,不含跨膜区域,存在信号肽序列,二级结构以随机卷曲和α-螺旋为主,无β-折叠,同源建模预测出牦牛TF蛋白与猪血清转铁蛋白有相似的三级结构,同源性可达71.91%。[结论]成功克隆了麦洼牦牛TF基因并分析了其蛋白质结构,为进一步研究TF蛋白功能提供理论基础。
文摘为了更好地了解市售椰浆中椰子蛋白质量状况,本研究以市售的10种椰浆为样品,采用反向高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)及十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium laurylsulfonate-polyacrylamide gel electrophoresis,SDS-PAGE)对提取蛋白进行分析,采用基质辅助激光解吸电离飞行时间串联质谱(matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry,MALDI-TOF/TOF-MS)对蛋白进行鉴定。结果表明:椰浆RP-HPLC约在10.4 min和11.9 min分别出现1个色谱峰,大多数样品峰面积大小都与蛋白标签含量一致,SDS-PAGE条带数量和颜色的深浅与RP-HPLC得到的峰面积结果一致,个别样品标签含量高,SDS-PAGE能分离出的蛋白条带数量也较多,但RP-HPLC的椰子蛋白峰面积小,经MALDI-TOF/TOF-MS鉴定,部分条带没有鉴定出椰子有关的肽段或蛋白。研究结果为椰浆蛋白的食品安全与质量控制提供了方法参考和示范。
基金support from the Program for Changjiang Scholars and Innovative Research Team in Universitythe Natioual High Technology Research and development Program of China (Grant No. 2007AA022007)+2 种基金Program for New Century Excellent Talents in University (Grant No. NCET-06-0697)the National Natural Science Foundation of China (Grant Nos. 90606003 & 20775021)Outstanding Youth Foundation of Hunan Province (Grant No. 08JJ10002)
文摘With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and biofunctionalized nanoparticles, a series of novel principles, methods and techniques have been exploited for bioanalytical and biomedical studies. This review mainly discusses the applications of molecular beacon probes and biofunctionalized nanoparticles-based technologies for realtime, in-situ, highly sensitive and highly selective protein analysis, including the nonspecific or specific protein detection and separation, protein/DNA interaction studies, cell surface protein recognition, and antigen-antibody binding process-based bacteria assays. The introduction of molecular beacon probes and biofunctionalized nanoparticles into the protein analysis area would necessarily advance the proteomics research.
文摘With the sequencing of human genome almost complete, human genome project enters the post-genome-sequencing era. Compared to genomics, the analysis of proteome is rather difficult since in human cells there are around 200000 proteins, which are expressed at any time at different levels of concentration with a wide dynamic range. In addition, a given proteome is not static but dynamic, being defined by a combination of the cell's genome, environment, and even its history. To meet the higher and higher requirements from proteome analysis, in our recent work, we have developed some capillary electrophoresis (CE)-based techniques for the analysis of complex protein samples.
