Recently a new cellulose acetate membrane for electrophoresis to analyze protein fractions, named SELECA-VSP, was developed by Toyo Roshi Kaisha Ltd. In advance of commercial supply, a study group was organized to investigate utility of this new membrane in comparison with SEPARAX-SP, a standard membrane for protein fraction assay widely used in Japan. As a member of this study group, our laboratory mainly investigated the difference between the two membranes when analyzing protein fractions in serum as well as ascites, pleural fluid, cerebrospinal fluid and urine. JOKO-CTE8000 was used as a protein fraction analyzer. When buffer lacking EDTA was used in electrophoresis of serum and ascites, the correlation efficiency between the two membranes in percentage of the β fraction was inferior to that of other fractions. When EDTA-containing buffer was used in analysis of serum and ascites, however, the correlation between the two membranes in β-fraction was improved to a comparable level to other fractions. The correlations between the two membranes were satisfactory in analysis of samples other than serum and ascites, regardless of the presence of EDTA in buffer. We conclude that this newly developed membrane has the similar performance to a standard membrane and could be applied in assay of protein fraction in clinical samples.
{"title":"全自動電気泳動装置（常光CTE8000）による臨床検査タンパク性能評価","authors":"京子 宮﨑, 正芳 米山, 美穂 高橋, 照夫 江上, 宏明 大西, 卓 渡邊","doi":"10.2198/sbk.51.113","DOIUrl":"https://doi.org/10.2198/sbk.51.113","url":null,"abstract":"Recently a new cellulose acetate membrane for electrophoresis to analyze protein fractions, named SELECA-VSP, was developed by Toyo Roshi Kaisha Ltd. In advance of commercial supply, a study group was organized to investigate utility of this new membrane in comparison with SEPARAX-SP, a standard membrane for protein fraction assay widely used in Japan. As a member of this study group, our laboratory mainly investigated the difference between the two membranes when analyzing protein fractions in serum as well as ascites, pleural fluid, cerebrospinal fluid and urine. JOKO-CTE8000 was used as a protein fraction analyzer. When buffer lacking EDTA was used in electrophoresis of serum and ascites, the correlation efficiency between the two membranes in percentage of the β fraction was inferior to that of other fractions. When EDTA-containing buffer was used in analysis of serum and ascites, however, the correlation between the two membranes in β-fraction was improved to a comparable level to other fractions. The correlations between the two membranes were satisfactory in analysis of samples other than serum and ascites, regardless of the presence of EDTA in buffer. We conclude that this newly developed membrane has the similar performance to a standard membrane and could be applied in assay of protein fraction in clinical samples.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"54 1","pages":"113-117"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78647240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Following the discontinuation of manufacturing SEPARAX-SP, a cellulose acetate membrane from Fujifilm which had long been used in clinical laboratory testing, Advantec Toyo decided to manufacture another cellulose acetate membrane that exhibits the properties of SEPARAX-SP. However, it is unclear whether SELECA-VSP, the new product based on certain changes in the manufacturing process of the cellulose acetate membrane, is equivalent to SEPARAX-SP in quality. Thus, we conducted a basic study using a prototype membrane of SELECA-VSP on an automatic electrophoresis apparatus, and found that it could be applied to the currently used model of automatic electrophoresis apparatus without adjusting operating conditions. The basic study results and their correlations were promising enough to suggest that SELECA-VSP could replace SEPARAX-SP without problems.
{"title":"新規膜「セレカ-VSP」を用いた血清タンパク分画の評価～全自動電気泳動分析装置CTE-1800（常光）による分析～","authors":"和代 中村, 秋恵 須郷, 栄治 宮島","doi":"10.2198/sbk.51.119","DOIUrl":"https://doi.org/10.2198/sbk.51.119","url":null,"abstract":"Following the discontinuation of manufacturing SEPARAX-SP, a cellulose acetate membrane from Fujifilm which had long been used in clinical laboratory testing, Advantec Toyo decided to manufacture another cellulose acetate membrane that exhibits the properties of SEPARAX-SP. However, it is unclear whether SELECA-VSP, the new product based on certain changes in the manufacturing process of the cellulose acetate membrane, is equivalent to SEPARAX-SP in quality. Thus, we conducted a basic study using a prototype membrane of SELECA-VSP on an automatic electrophoresis apparatus, and found that it could be applied to the currently used model of automatic electrophoresis apparatus without adjusting operating conditions. The basic study results and their correlations were promising enough to suggest that SELECA-VSP could replace SEPARAX-SP without problems.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"606 1","pages":"119-123"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77464417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-12-01DOI: 10.2198/JELECTROPH.50.33
Yoko Ino, Akiko Okayama, Y. Iwafune, Noriaki Arakawa, J. Kikuchi, M. Kamita, H. Kawasaki, Takeshi Okada, H. Hirano
We have developed a technique, which can produce high-density protein chips. In this technique, proteins are separated by gel electrophoresis, and electroblotted by semidry blotting apparatus onto a diamond-like carbon-coated stainless-steel plate (DLC plate) of which surface is modified chemically with amino groups. Proteins immobilized on this protein chip can interact with other proteins in solution, and proteins interacted with the immobilized proteins can be identified by mass spectrometric analysis. However, the electroblotting efficiency was not stable. We anticipated that the unstable efficiency might be related to the fluctuation of temperature during electroblotting. In the present study, to investigate the optimal temperature for efficient and effective electroblotting, we developed a semidry blotting apparatus, which could regulate blotting temperature from 4 to 45°C (±1°C). The high and reproducible blotting efficiency was obtained at not low temperature but rather high temperature (30°C). The temperature did not impede protein identification or analysis of protein-protein interactions on the DLC plates by mass spectrometry.
{"title":"Efficient electroblotting of gel-resolved proteins onto diamond-like carbon-coated plate for protein-chip","authors":"Yoko Ino, Akiko Okayama, Y. Iwafune, Noriaki Arakawa, J. Kikuchi, M. Kamita, H. Kawasaki, Takeshi Okada, H. Hirano","doi":"10.2198/JELECTROPH.50.33","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.33","url":null,"abstract":"We have developed a technique, which can produce high-density protein chips. In this technique, proteins are separated by gel electrophoresis, and electroblotted by semidry blotting apparatus onto a diamond-like carbon-coated stainless-steel plate (DLC plate) of which surface is modified chemically with amino groups. Proteins immobilized on this protein chip can interact with other proteins in solution, and proteins interacted with the immobilized proteins can be identified by mass spectrometric analysis. However, the electroblotting efficiency was not stable. We anticipated that the unstable efficiency might be related to the fluctuation of temperature during electroblotting. In the present study, to investigate the optimal temperature for efficient and effective electroblotting, we developed a semidry blotting apparatus, which could regulate blotting temperature from 4 to 45°C (±1°C). The high and reproducible blotting efficiency was obtained at not low temperature but rather high temperature (30°C). The temperature did not impede protein identification or analysis of protein-protein interactions on the DLC plates by mass spectrometry.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"10 1","pages":"33-37"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87014386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-12-01DOI: 10.2198/JELECTROPH.50.39
H. Kajiwara, K. Nakane, Jiang Piyang, A. Imamaki, Yoko Ito, Fumio Togasaki, Tsuyoshi Kotake, Hikari Murai, Masatoshi Nakamura, K. Mita, R. Nomura, Yuji Shimizu, Michihiko Shimomura, M. Ishizaka
A silkworm (Bombyx mori) proteome database (SPD) was constructed using Make-2DDB II software. The present SPD contains the proteomes of the seven major tissues in the silkworm: the middle silkglands, posterior silkglands, midguts, fat bodies, hemolymph, ovaries, and Malpighian tubules. Proteins in each tissue at day 3 of the fifth instar were identified by MS/MS analysis and the protein profiles by 2-DE from day 1 of the fourth instar larva to the adult moth were constructed.
{"title":"Draft of silkworm proteome database","authors":"H. Kajiwara, K. Nakane, Jiang Piyang, A. Imamaki, Yoko Ito, Fumio Togasaki, Tsuyoshi Kotake, Hikari Murai, Masatoshi Nakamura, K. Mita, R. Nomura, Yuji Shimizu, Michihiko Shimomura, M. Ishizaka","doi":"10.2198/JELECTROPH.50.39","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.39","url":null,"abstract":"A silkworm (Bombyx mori) proteome database (SPD) was constructed using Make-2DDB II software. The present SPD contains the proteomes of the seven major tissues in the silkworm: the middle silkglands, posterior silkglands, midguts, fat bodies, hemolymph, ovaries, and Malpighian tubules. Proteins in each tissue at day 3 of the fifth instar were identified by MS/MS analysis and the protein profiles by 2-DE from day 1 of the fourth instar larva to the adult moth were constructed.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"50 1","pages":"39-41"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89600165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-12-01DOI: 10.2198/JELECTROPH.50.25
K. Sakaguchi, J. Suzuki, Masaki Tanaka, M. Shirai, F. Akahori
Polychlorinated biphenyl (PCB) 126 or PCB 169 was administered to pregnant rats and kinetic changes in plasma protein spots of their next generation (F2 rats) ages 1, 3, 6, and 15 weeks were observed by micro two-dimensional polyacrylamide gel electrophoresis (M2D-PAGE). In the control group in which age-related changes were observable, changes in the form of the albumin (Alb) spot were recognized at ages 1-3 weeks. Only the Alb spot on the acid side, i.e., non-mercaptoalbumin, was recognized at age 1 week. At age 3 weeks, the Alb spot on the alkaline side, i.e., mercaptoalbumin, also became observable. On the other hand, qualitative changes in the form of Alb, i.e., manifestations of mercaptoalbumin, were recognized at age 1 week by administration of PCB 126. This result suggested that the influence of PCB 126 on the living body is greater than that of PCB 169. In spite of the treatment for toxicity by equivalence of dose control on the basis of toxicity equivalency factor (TEF) 0.1 for PCB 126 (PeCB) and 0.01 for PCB 169 (HxCB), the influence of PCB 126 on the living body was greater than that of PCB 169. The C3 spot was increased at age 3 weeks in the PCB 126 and the PCB 169. The spot showed a tendency toward increase at age 6 weeks in the PCB 126. The TEF compared PCB169 with PCB126, and distinctly increase of the C3 spot was observed for 15 weeks though the TEF was low. From these, as for the PCB 169, it was suggested appearing remarkably in aging influence of the immunity toxicity. The observations of the changes in these spots led to the conclusion that the changes in these spots resulted from transfer of the drugs accumulating in the maternal body to the F2 rats of the maternal rats administered PCB 126 and those administered PCB 169 via the breast milk and that these changes are involved with inflammatory proteins, including the target genes influenced by the toxicity of coplanar PCBs.
{"title":"Micro two-dimensional electrophoretic analysis of changes in plasma proteins of next-generation rat of the pregnant rats administered to coplanar PCBs","authors":"K. Sakaguchi, J. Suzuki, Masaki Tanaka, M. Shirai, F. Akahori","doi":"10.2198/JELECTROPH.50.25","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.25","url":null,"abstract":"Polychlorinated biphenyl (PCB) 126 or PCB 169 was administered to pregnant rats and kinetic changes in plasma protein spots of their next generation (F2 rats) ages 1, 3, 6, and 15 weeks were observed by micro two-dimensional polyacrylamide gel electrophoresis (M2D-PAGE). In the control group in which age-related changes were observable, changes in the form of the albumin (Alb) spot were recognized at ages 1-3 weeks. Only the Alb spot on the acid side, i.e., non-mercaptoalbumin, was recognized at age 1 week. At age 3 weeks, the Alb spot on the alkaline side, i.e., mercaptoalbumin, also became observable. On the other hand, qualitative changes in the form of Alb, i.e., manifestations of mercaptoalbumin, were recognized at age 1 week by administration of PCB 126. This result suggested that the influence of PCB 126 on the living body is greater than that of PCB 169. In spite of the treatment for toxicity by equivalence of dose control on the basis of toxicity equivalency factor (TEF) 0.1 for PCB 126 (PeCB) and 0.01 for PCB 169 (HxCB), the influence of PCB 126 on the living body was greater than that of PCB 169. The C3 spot was increased at age 3 weeks in the PCB 126 and the PCB 169. The spot showed a tendency toward increase at age 6 weeks in the PCB 126. The TEF compared PCB169 with PCB126, and distinctly increase of the C3 spot was observed for 15 weeks though the TEF was low. From these, as for the PCB 169, it was suggested appearing remarkably in aging influence of the immunity toxicity. The observations of the changes in these spots led to the conclusion that the changes in these spots resulted from transfer of the drugs accumulating in the maternal body to the F2 rats of the maternal rats administered PCB 126 and those administered PCB 169 via the breast milk and that these changes are involved with inflammatory proteins, including the target genes influenced by the toxicity of coplanar PCBs.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"67 1","pages":"25-31"},"PeriodicalIF":0.0,"publicationDate":"2006-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83255791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-06-01DOI: 10.2198/JELECTROPH.50.19
K. Fujita, F. Kameko, Y. Kato, M. Fukushima, N. Okumura, F. Terasawa, M. Sugano, K. Yamauchi, Hirohisa Sato, M. Kameko, I. Sakurabayashi
We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the mechanism of IgA-albumin complex formation was not clear. We isolated the IgA-albumin complexes from 5 IgA type M-proteinemia patients’ sera. To elucidate the mechanism of IgA-albumin complex formation, we performed the dissociation assay of IgA-albumin complexes, the identification of albumin binding sites of monoclonal IgA using immunoelectrophoresis, western blotting and chromatography technologies. In all patients with IgA type M-proteinemia, the IgA-albumin complexes were dissociated by treated with 2-mercaptoethanol (2-ME), but not by treated with a strong acid as acetic acid or NaCl of high concentrations. Moreover, when the purified monoclonal IgA containing IgA-albumin complexes was digested with the IgA protease from Neisseria gonorrhoeae, no macro-albumin was demonstrated. It seems probable that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the binding site of albumin is located in near the hinge region of IgA molecule and involve the free SH group thought to be present in the α-chain.
{"title":"Mechanism of IgA-albumin complex formation that affects the fructosamine assay","authors":"K. Fujita, F. Kameko, Y. Kato, M. Fukushima, N. Okumura, F. Terasawa, M. Sugano, K. Yamauchi, Hirohisa Sato, M. Kameko, I. Sakurabayashi","doi":"10.2198/JELECTROPH.50.19","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.19","url":null,"abstract":"We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the mechanism of IgA-albumin complex formation was not clear. We isolated the IgA-albumin complexes from 5 IgA type M-proteinemia patients’ sera. To elucidate the mechanism of IgA-albumin complex formation, we performed the dissociation assay of IgA-albumin complexes, the identification of albumin binding sites of monoclonal IgA using immunoelectrophoresis, western blotting and chromatography technologies. In all patients with IgA type M-proteinemia, the IgA-albumin complexes were dissociated by treated with 2-mercaptoethanol (2-ME), but not by treated with a strong acid as acetic acid or NaCl of high concentrations. Moreover, when the purified monoclonal IgA containing IgA-albumin complexes was digested with the IgA protease from Neisseria gonorrhoeae, no macro-albumin was demonstrated. It seems probable that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the binding site of albumin is located in near the hinge region of IgA molecule and involve the free SH group thought to be present in the α-chain.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"269 1","pages":"19-23"},"PeriodicalIF":0.0,"publicationDate":"2006-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76953389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-06-01DOI: 10.2198/JELECTROPH.50.13
Xiulian Zhang, Y. Kuramitsu, M. Fujimoto, K. Nakamura
Studies on blood plasma proteome were performed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) using samples supplied from China and the United States by collaborations of Human Plasma Proteome Project of HUPO. The plasma proteins were separated by isoelectric focusing using capillary type polyacrylamide gel (PAG) with carrier ampholytes pH 3.5-10 in the first dimension and with SDS-PAGE using 15%, 12.5% and 8% gel in the second dimension. The protein spots were identified by peptide mass fingerprinting using electrospray ionization MS/MS (LC/MSD Trap XCT, Agilent). More than 145 protein spots were identified by LC-MS/MS. The 2-DE patterns showed that the samples stored at 4°C for 3 days lost inter-alpha-trypsin inhibitor heavy chain 4 (ITIH-4).
{"title":"Comparative 2-DE proteomic analysis clarified that the stability of ITIH-4 is decreased under the storage at 4°C","authors":"Xiulian Zhang, Y. Kuramitsu, M. Fujimoto, K. Nakamura","doi":"10.2198/JELECTROPH.50.13","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.13","url":null,"abstract":"Studies on blood plasma proteome were performed by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) using samples supplied from China and the United States by collaborations of Human Plasma Proteome Project of HUPO. The plasma proteins were separated by isoelectric focusing using capillary type polyacrylamide gel (PAG) with carrier ampholytes pH 3.5-10 in the first dimension and with SDS-PAGE using 15%, 12.5% and 8% gel in the second dimension. The protein spots were identified by peptide mass fingerprinting using electrospray ionization MS/MS (LC/MSD Trap XCT, Agilent). More than 145 protein spots were identified by LC-MS/MS. The 2-DE patterns showed that the samples stored at 4°C for 3 days lost inter-alpha-trypsin inhibitor heavy chain 4 (ITIH-4).","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"31 1","pages":"13-17"},"PeriodicalIF":0.0,"publicationDate":"2006-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77616688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We modified the silver staining method of Kerenyi and Gallyas (Clin Chim Acta 1972; 38: 465-467) for visualizing low levels of protein with our own agarose gels as reported previously (Yokomizo K. et al. J Electrophoresis 2003; 47: 91-97). To reduce background staining and avoid the formation of artifact spots, a number of factors were studied. These included the composition of the staining reagent and fixative solutions (first and second fixation). It was found that 2.5% Na2CO3, 0.1% AgNO3, 0.1% NH4NO3, 0.75% tungtosilicic acid and 0.14% formaldehyde of the staining reagent mixture was critical and that the addition of 5% ZnSO4 to the second fixative solution resulted in consistently lower background staining and a significant reduction in artifact spots. Our silver staining procedure is approximately 100-fold more sensitive than Coomassie brilliant blue and the detection limit of bovine serum albumin is about 1.46 ng per band.
我们改进了Kerenyi和Gallyas的银染色方法(临床化学学报1972;38: 465-467)，用我们自己的琼脂糖凝胶可视化低水平的蛋白质，正如之前报道的那样(Yokomizo K. et al.)。J Electrophoresis 2003;47: 91 - 97)。为了减少背景染色和避免伪斑的形成，研究了许多因素。这些包括染色试剂和固定溶液的组成(第一次和第二次固定)。结果发现，2.5% Na2CO3、0.1% AgNO3、0.1% NH4NO3、0.75%钨硅酸和0.14%甲醛是染色试剂混合物的临界浓度，在第二次固定液中添加5% ZnSO4可使背景染色持续降低，并显著减少伪影斑点。我们的银染色方法的灵敏度比考马斯亮蓝高约100倍，牛血清白蛋白的检出限约为每波段1.46 ng。
{"title":"A silver staining procedure for proteins in agarose gels","authors":"K. Yokomizo, S. Maruya, N. Hiratsuka, K. Shiba","doi":"10.2198/JELECTROPH.50.1","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.1","url":null,"abstract":"We modified the silver staining method of Kerenyi and Gallyas (Clin Chim Acta 1972; 38: 465-467) for visualizing low levels of protein with our own agarose gels as reported previously (Yokomizo K. et al. J Electrophoresis 2003; 47: 91-97). To reduce background staining and avoid the formation of artifact spots, a number of factors were studied. These included the composition of the staining reagent and fixative solutions (first and second fixation). It was found that 2.5% Na2CO3, 0.1% AgNO3, 0.1% NH4NO3, 0.75% tungtosilicic acid and 0.14% formaldehyde of the staining reagent mixture was critical and that the addition of 5% ZnSO4 to the second fixative solution resulted in consistently lower background staining and a significant reduction in artifact spots. Our silver staining procedure is approximately 100-fold more sensitive than Coomassie brilliant blue and the detection limit of bovine serum albumin is about 1.46 ng per band.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"18 1","pages":"1-5"},"PeriodicalIF":0.0,"publicationDate":"2006-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80538524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The transplanted GFP-positive BMCs (especially the Liv8 negative cell population, without culturing) migrated into the peri-portal regions of the cirrhotic liver. They differentiated into Liv2-positive hepatoblasts and then into albumin-producing hepatocytes. The differentiation “niche” induced by persistent liver damage due to continuous CCl4 injection seems to be an essential factor. Microarry-SOM analysis showed that at an early stage after BMC transplantation, the genes related to morphology were activated. Then later, genes associated with liver metabolism were activated. Finally, BMC transplantation improved liver function, liver fibrosis and the survival rate. These findings strongly support the development of a new cell therapy using autologous BMCs to treat liver cirrhosis patients, because BMC transplantation itself is an established treatment for hematological diseases. Our finding indicates that FGF2 will accelerate the differentiation of BMC to hepatocyte.Based on the results obtained in basic research using the GFP/CCl4 model, human trials are now undergoing.
{"title":"Cell therapy with bone marrow cell for liver cirrhosis","authors":"I. Sakaida","doi":"10.2198/JELECTROPH.50.7","DOIUrl":"https://doi.org/10.2198/JELECTROPH.50.7","url":null,"abstract":"The transplanted GFP-positive BMCs (especially the Liv8 negative cell population, without culturing) migrated into the peri-portal regions of the cirrhotic liver. They differentiated into Liv2-positive hepatoblasts and then into albumin-producing hepatocytes. The differentiation “niche” induced by persistent liver damage due to continuous CCl4 injection seems to be an essential factor. Microarry-SOM analysis showed that at an early stage after BMC transplantation, the genes related to morphology were activated. Then later, genes associated with liver metabolism were activated. Finally, BMC transplantation improved liver function, liver fibrosis and the survival rate. These findings strongly support the development of a new cell therapy using autologous BMCs to treat liver cirrhosis patients, because BMC transplantation itself is an established treatment for hematological diseases. Our finding indicates that FGF2 will accelerate the differentiation of BMC to hepatocyte.Based on the results obtained in basic research using the GFP/CCl4 model, human trials are now undergoing.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"8 1","pages":"7-12"},"PeriodicalIF":0.0,"publicationDate":"2006-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90096042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-12-01DOI: 10.2198/JELECTROPH.49.77
Megumi Nakamura, Y. Sakurai, Yasuo Takeda, T. Toda
There is increasing evidence that lipid rafts may play important roles in brain neuronal cell functions including signal transduction. Meanwhile, the results suggesting possible presence of rafts in intracellular organelles such as mitochondria have been also reported. In this study, we compared proteins in raft-like structure of mitochondria to those of synaptosomal rafts and analyzed age-related alterations in protein in both mitochondrial and synaptosomal lipid rafts. A low density Triton X-100-insoluble fraction was prepared from cerebral cortical synaptosome and mitochondria of mouse and analyzed by two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry. Co-localization of flotillin and cholesterol in Triton X-100-insoluble fraction of mitochondria was shown by Western blot analysis. Differential display of proteins using computer-aided image analysis revealed that the composition of protein in flotillin-rich Triton X-100-insoluble fraction of mitochondria was similar to that found in synaptosome. Several protein spots on 2-D gels varied in quantity depending on the age of the mouse, including the guanine nucleotide-binding protein G(O) alpha subunit, as identified by peptide mass fingerprinting.
越来越多的证据表明，脂筏可能在脑神经细胞的信号转导等功能中发挥重要作用。同时，研究结果表明，筏可能存在于细胞内细胞器，如线粒体。在这项研究中，我们比较了线粒体筏状结构和突触体筏状结构中的蛋白质，并分析了线粒体和突触体脂筏中蛋白质的年龄相关变化。从小鼠大脑皮层突触体和线粒体制备低密度Triton x -100不溶性组分，采用二维凝胶电泳结合质谱分析。Western blot分析显示，线粒体Triton x -100不溶性部分存在flotillin和胆固醇的共定位。利用计算机辅助图像分析的蛋白质差异显示显示，线粒体中富含浮胞蛋白的Triton x -100不溶性部分的蛋白质组成与突触体中的蛋白质组成相似。2d凝胶上的几个蛋白点的数量根据小鼠的年龄而变化，包括鸟嘌呤核苷酸结合蛋白G(O) α亚基，通过肽质量指纹图谱识别。
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