An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported.The main improvement was using a fixing step of 25% trichloroacetic acid(TCA) before CBB staining.For most proteins s...An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported.The main improvement was using a fixing step of 25% trichloroacetic acid(TCA) before CBB staining.For most proteins studied,the sensitivity of the improved CBB staining was about twice as high as that of the typical method.For basic and low molecular weight proteins such as ribosomal proteins,the sensitivity of this improved staining method was about 3.5-28 times that of the typical method.It was speculated that the improved procedure would be suitable for exact quantitative analysis of proteins fractionated by SDS-PAGE,especially for basic and low molecular weight proteins.On the other hand,this new modified method might be also applied to multidisciplinary studies,such as biological researches and nuclear sciences.展开更多
为建立一套适合海滨木槿叶片蛋白质组学分析的双向电泳体系,以海滨木槿无性系叶片为材料,分别采用裂解液法、三氯乙酸-丙酮沉淀法、改良酚抽法分离纯化蛋白质,选用24 cm pH 3～10的IPG胶条,并对不同上样量、等电聚焦程序、平衡时间等电...为建立一套适合海滨木槿叶片蛋白质组学分析的双向电泳体系,以海滨木槿无性系叶片为材料,分别采用裂解液法、三氯乙酸-丙酮沉淀法、改良酚抽法分离纯化蛋白质,选用24 cm pH 3～10的IPG胶条,并对不同上样量、等电聚焦程序、平衡时间等电泳条件进行优化。结果表明:采用改良酚抽法提取蛋白质,上样量为每IPG胶条1 000μg,总聚焦功率90 kV.h,平衡时间15 min,胶体考马斯亮蓝染色,可得到蛋白质点数目多、分辨率高的双向电泳图谱。重复性试验结果显示,该体系具有很好的稳定性及可重复性,可满足海滨木槿蛋白质组学研究的要求。展开更多
Antarctic ice microalgae Chlamydomonas sp.ICE-L can survive and thrive in Antarctic sea ice.In this study,Chlamydomonas sp.ICE-L could survive at the salinity of 132‰ NaCl.SDS-PAGE showed that the density of 2 bands...Antarctic ice microalgae Chlamydomonas sp.ICE-L can survive and thrive in Antarctic sea ice.In this study,Chlamydomonas sp.ICE-L could survive at the salinity of 132‰ NaCl.SDS-PAGE showed that the density of 2 bands（26 and 36 kD） decreased obviously at the salinity of 99‰ NaCl compared to at the salinity of 33‰ NaCl.The soluble proteins in Chlamydomonas sp.ICE-L grown under salinity of 33‰ and 99% NaCl were compared by 2-D gel electrophoresis.After shocking with high salinity,8 protein spots were found to disappear,and the density of 28 protein spots decreased.In addition,19 protein spots were enhanced or induced,including one new peptide（51 kD）.The changes of proteins might be correlated with the resistance for Chlamydomonas sp.ICE-L to high salinity.展开更多
Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-d...Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.展开更多
Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgeni...Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.展开更多
文摘An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported.The main improvement was using a fixing step of 25% trichloroacetic acid(TCA) before CBB staining.For most proteins studied,the sensitivity of the improved CBB staining was about twice as high as that of the typical method.For basic and low molecular weight proteins such as ribosomal proteins,the sensitivity of this improved staining method was about 3.5-28 times that of the typical method.It was speculated that the improved procedure would be suitable for exact quantitative analysis of proteins fractionated by SDS-PAGE,especially for basic and low molecular weight proteins.On the other hand,this new modified method might be also applied to multidisciplinary studies,such as biological researches and nuclear sciences.
基金supported by the National Natural Science Foundation of China(No.40876107No.40876102)Marine Science Foundation for Young Scientists of the State Oceanic Administration(2010122)
文摘Antarctic ice microalgae Chlamydomonas sp.ICE-L can survive and thrive in Antarctic sea ice.In this study,Chlamydomonas sp.ICE-L could survive at the salinity of 132‰ NaCl.SDS-PAGE showed that the density of 2 bands（26 and 36 kD） decreased obviously at the salinity of 99‰ NaCl compared to at the salinity of 33‰ NaCl.The soluble proteins in Chlamydomonas sp.ICE-L grown under salinity of 33‰ and 99% NaCl were compared by 2-D gel electrophoresis.After shocking with high salinity,8 protein spots were found to disappear,and the density of 28 protein spots decreased.In addition,19 protein spots were enhanced or induced,including one new peptide（51 kD）.The changes of proteins might be correlated with the resistance for Chlamydomonas sp.ICE-L to high salinity.
基金This work was supported by a grant from National 973 Project (2001CB5102) for Outstanding Scholars of New Era from the Ministry of Education of China (2002-48)+1 种基金 National Natural Science Foundation of China (30000028) the key research program from Sc
文摘Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.
基金supported by the National Priority Basic Research Programs of People’s Republic of China: Bio-safety Study on GMOs of Agricultural Importance (GN 2001CB109002)
文摘Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.
