Yooksil Sin, T. Ono, R. Tsuchiya, Rei Noguchi, Yuki Yoshimatsu, H. Kosako, T. Kondo
{"title":"Proteomic analysis of spheroids of rhabdomyosarcoma cells cultured with decellularized muscle extracts","authors":"Yooksil Sin, T. Ono, R. Tsuchiya, Rei Noguchi, Yuki Yoshimatsu, H. Kosako, T. Kondo","doi":"10.2198/jelectroph.66.1","DOIUrl":"https://doi.org/10.2198/jelectroph.66.1","url":null,"abstract":"","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"32 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87467295","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 : 2022-01-01DOI: 10.2198/jelectroph.66.13
Emiko Kinoshita-Kikuta, Yoko Ino, Y. Kimura, Tomoko Akiyama, E. Kinoshita, T. Koike
{"title":"Use of Escherichia coli expression system for analyzing kinase motifs","authors":"Emiko Kinoshita-Kikuta, Yoko Ino, Y. Kimura, Tomoko Akiyama, E. Kinoshita, T. Koike","doi":"10.2198/jelectroph.66.13","DOIUrl":"https://doi.org/10.2198/jelectroph.66.13","url":null,"abstract":"","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"100 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83607262","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}
{"title":"Electrophoretic extraction of protein complexes after separation and detection by a combined method of non-denaturing two-dimensional electrophoresis and reversible staining","authors":"Karin Nakao, Y. Shimazaki","doi":"10.2198/jelectroph.66.5","DOIUrl":"https://doi.org/10.2198/jelectroph.66.5","url":null,"abstract":"","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79200188","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}
Rei Noguchi, Yuki Yoshimatsu, T. Ono, Akane Sei, T. Kondo
Regulation of kinase activity plays a crucial role in carcinogenesis and cancer progression. Mutations in the activity domain of kinases are extensively investigated as therapeutic targets. We examined anti-proliferative anti-cancer drugs and drug targets via the multi-omics approach: (i) comprehensive kinase activity assay, (ii) high-throughput drug screening, and (iii) genomic sequencing. Two osteosarcomas cell lines, NCC-OS1-C1 and NCC-ESOS1-C1 derived from bone and soft tissue respectively, were used. Genetic alterations were examined by NCC Oncopanel based on the next-generation sequencing technology and SNP array. One hundred kinases were monitored by the PamStation 12, an in vitro kinase assay. The anti-proliferative effects of 214 FDA-approved anti-cancer drugs were examined. Mutation of PIK3CA and deletion of CDKN2A were identified in NCC-ESOS1-C1 and druggable genetic alterations were not identified in the NCC-OS1-C1. PI3K-AKT pathway or CDKN2A inhibitors did not show significant effects on these cell lines. Comprehensive kinomic assay revealed no remarkable differences on these osteosarcoma cells (R2=0.99). The two cells shared similar kinase activity profiles for FES, FER, PDGFR-β, VEGFR2, and Wee1. Anti-proliferative effects of anti-cancer drugs on NCC-OS1-C1 and NCC-ESOS1 cells showed remarkable differences. Significant responses to romidepsin and trabectedin were observed for both. Eribulin was effective on NCCOS1-C1; ifosfamide and dacarbazine were effective on NCC-ESOS1-C1 only. Hence, investigating kinase activities and genetic alterations will lead to predict the effects of kinase inhibitors. The different status of kinase mutations, activities, and response to inhibitors should be integrated. Multi-omics experiments and data integration are crucial in understanding cancer progression and developing novel therapies.
{"title":"Proteogenomic approach to drug targets in osteosarcomas with different original sites","authors":"Rei Noguchi, Yuki Yoshimatsu, T. Ono, Akane Sei, T. Kondo","doi":"10.2198/JELECTROPH.65.1","DOIUrl":"https://doi.org/10.2198/JELECTROPH.65.1","url":null,"abstract":"Regulation of kinase activity plays a crucial role in carcinogenesis and cancer progression. Mutations in the activity domain of kinases are extensively investigated as therapeutic targets. We examined anti-proliferative anti-cancer drugs and drug targets via the multi-omics approach: (i) comprehensive kinase activity assay, (ii) high-throughput drug screening, and (iii) genomic sequencing. Two osteosarcomas cell lines, NCC-OS1-C1 and NCC-ESOS1-C1 derived from bone and soft tissue respectively, were used. Genetic alterations were examined by NCC Oncopanel based on the next-generation sequencing technology and SNP array. One hundred kinases were monitored by the PamStation 12, an in vitro kinase assay. The anti-proliferative effects of 214 FDA-approved anti-cancer drugs were examined. Mutation of PIK3CA and deletion of CDKN2A were identified in NCC-ESOS1-C1 and druggable genetic alterations were not identified in the NCC-OS1-C1. PI3K-AKT pathway or CDKN2A inhibitors did not show significant effects on these cell lines. Comprehensive kinomic assay revealed no remarkable differences on these osteosarcoma cells (R2=0.99). The two cells shared similar kinase activity profiles for FES, FER, PDGFR-β, VEGFR2, and Wee1. Anti-proliferative effects of anti-cancer drugs on NCC-OS1-C1 and NCC-ESOS1 cells showed remarkable differences. Significant responses to romidepsin and trabectedin were observed for both. Eribulin was effective on NCCOS1-C1; ifosfamide and dacarbazine were effective on NCC-ESOS1-C1 only. Hence, investigating kinase activities and genetic alterations will lead to predict the effects of kinase inhibitors. The different status of kinase mutations, activities, and response to inhibitors should be integrated. Multi-omics experiments and data integration are crucial in understanding cancer progression and developing novel therapies.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90419237","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 : 2021-01-01DOI: 10.2198/JELECTROPH.65.13
R. Tsuchiya, Yuki Yoshimatsu, Naoto Tsuchiya, S. Ohtori, A. Kawai, T. Kondo
Sarcoma is a rare mesenchymal malignancy that comprises more than 50 histological subtypes. Because of the rarity and diversity of sarcomas, their differential diagnosis is difficult, and there is still a need for biomarkers to support pathological diagnoses. Micro RNAs (miRNAs) are small noncoding RNAs that regulate the behavior of tumors, such as invasion and metastasis. The expression patterns of miRNAs reflect the origin of malignancy and are considered to be candidate biomarkers. To understand the molecular background of those histological subtypes, we investigated the miRNA expression in 89 tumor tissues of eight subtypes. The correlation coefficients between each sarcoma subtype on the basis of miRNA expression values were mostly higher than 0.7, reflecting the common mesenchymal origin. By contrast, hierarchical clustering and principal component analysis showed that three types of sarcoma with chromosomal translocation (i.e., dermatofibrosarcoma protuberans, myxoid liposarcoma, and synovial sarcoma) were grouped according to their histological subtypes, whereas five types with complex karyotypes (i.e., myxofibrosarcoma, malignant peripheral nerve sheath tumor, undifferentiated pleomorphic sarcoma, dedifferentiated liposarcoma, and pleomorphic liposarcoma) were not. Notably, the number of miRNAs whose expression pattern was unique to histological subtypes with statistical significance was higher in sarcomas with chromosome translocation than in those with complex karyotypes. Hence, it can be concluded that the miRNAs unique to histological subtypes are candidate biomarkers for the differential diagnosis of sarcomas, particularly in those with chromosomal translocation.
{"title":"Comprehensive miRNA expression analysis for histological subtypes of soft tissue sarcoma","authors":"R. Tsuchiya, Yuki Yoshimatsu, Naoto Tsuchiya, S. Ohtori, A. Kawai, T. Kondo","doi":"10.2198/JELECTROPH.65.13","DOIUrl":"https://doi.org/10.2198/JELECTROPH.65.13","url":null,"abstract":"Sarcoma is a rare mesenchymal malignancy that comprises more than 50 histological subtypes. Because of the rarity and diversity of sarcomas, their differential diagnosis is difficult, and there is still a need for biomarkers to support pathological diagnoses. Micro RNAs (miRNAs) are small noncoding RNAs that regulate the behavior of tumors, such as invasion and metastasis. The expression patterns of miRNAs reflect the origin of malignancy and are considered to be candidate biomarkers. To understand the molecular background of those histological subtypes, we investigated the miRNA expression in 89 tumor tissues of eight subtypes. The correlation coefficients between each sarcoma subtype on the basis of miRNA expression values were mostly higher than 0.7, reflecting the common mesenchymal origin. By contrast, hierarchical clustering and principal component analysis showed that three types of sarcoma with chromosomal translocation (i.e., dermatofibrosarcoma protuberans, myxoid liposarcoma, and synovial sarcoma) were grouped according to their histological subtypes, whereas five types with complex karyotypes (i.e., myxofibrosarcoma, malignant peripheral nerve sheath tumor, undifferentiated pleomorphic sarcoma, dedifferentiated liposarcoma, and pleomorphic liposarcoma) were not. Notably, the number of miRNAs whose expression pattern was unique to histological subtypes with statistical significance was higher in sarcomas with chromosome translocation than in those with complex karyotypes. Hence, it can be concluded that the miRNAs unique to histological subtypes are candidate biomarkers for the differential diagnosis of sarcomas, particularly in those with chromosomal translocation.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"122 17","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91403052","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 : 2021-01-01DOI: 10.2198/jelectroph.65.39
H. Kajiwara, R. Murakami, K. Nakajima
SUMMARY A method for the analysis of silkworm ( Bombyx mori L.) cocoons and silk fibers was established using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method was evaluated on the most popu-lar four-way cross hybrid race Kinshu × Showa and its parent races Kinshu and Showa . Most of the peaks observed in the peptide mass fingerprints of the Kinshu × Showa cocoon were from the parent races Kinshu and Showa . Simultaneous acid cleavage of silk fibers at room temperature is a characteristic phenomenon that has not been observed in spider silk and animal hair.
{"title":"Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based biotyping of silk. I. Method development","authors":"H. Kajiwara, R. Murakami, K. Nakajima","doi":"10.2198/jelectroph.65.39","DOIUrl":"https://doi.org/10.2198/jelectroph.65.39","url":null,"abstract":"SUMMARY A method for the analysis of silkworm ( Bombyx mori L.) cocoons and silk fibers was established using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method was evaluated on the most popu-lar four-way cross hybrid race Kinshu × Showa and its parent races Kinshu and Showa . Most of the peaks observed in the peptide mass fingerprints of the Kinshu × Showa cocoon were from the parent races Kinshu and Showa . Simultaneous acid cleavage of silk fibers at room temperature is a characteristic phenomenon that has not been observed in spider silk and animal hair.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91005056","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 : 2021-01-01DOI: 10.2198/jelectroph.65.23
Rei Noguchi, Yuki Yoshimatsu, Akane Sei, H. Yoshida, Tomoyasu Katou, T. Kondo
Clear cell carcinoma (CCC) is a rare subtype of ovarian cancer resistant to standard platinum chemotherapy, which leads to a poor prognosis for patients with CCC. Kinases are targets for anticancer drugs; few studies have profiled kinase activity to identify kinase inhibitors as novel anticancer drugs. In this study, we aimed to identify novel anticancer drugs for the treatment of CCC with comprehensive kinase activity assay and drug screening. Using ascites from a 51-year old patient, we established and characterized the NCC-cOV1-C1 cell line. We screened the antiproliferative effects of 152 small anticancer compounds and conducted comprehensive kinase activity assays with the PamStation12 platform. The NCC-cOV1-C1 cells harbor copy number variation of HFN1β amplification, and exhibit constant growth, spheroid formation, and invasion capability. NCC-cOV1-C1 cells responded remarkably to idarubicin HCl and vorinostat. The kinase activity assay revealed that SRC and EGFR were highly activated in NCC-cOV1-C1 cells; the SRC inhibitor dasatinib and the EGFR inhibitor lapatinib exhibited antiproliferative effects and down-regulation of downstream signaling. The NCC-cOV1-C1 cell line will be a useful tool for basic and preclinical study of CCC, and the clinical utility of idarubicin HCl, vorinostat, dasatinib, lapatinib is worthy of further investigation.
{"title":"Drug screening and kinase activity profiling of a novel patient-derived cell line of clear cell ovarian carcinoma","authors":"Rei Noguchi, Yuki Yoshimatsu, Akane Sei, H. Yoshida, Tomoyasu Katou, T. Kondo","doi":"10.2198/jelectroph.65.23","DOIUrl":"https://doi.org/10.2198/jelectroph.65.23","url":null,"abstract":"Clear cell carcinoma (CCC) is a rare subtype of ovarian cancer resistant to standard platinum chemotherapy, which leads to a poor prognosis for patients with CCC. Kinases are targets for anticancer drugs; few studies have profiled kinase activity to identify kinase inhibitors as novel anticancer drugs. In this study, we aimed to identify novel anticancer drugs for the treatment of CCC with comprehensive kinase activity assay and drug screening. Using ascites from a 51-year old patient, we established and characterized the NCC-cOV1-C1 cell line. We screened the antiproliferative effects of 152 small anticancer compounds and conducted comprehensive kinase activity assays with the PamStation12 platform. The NCC-cOV1-C1 cells harbor copy number variation of HFN1β amplification, and exhibit constant growth, spheroid formation, and invasion capability. NCC-cOV1-C1 cells responded remarkably to idarubicin HCl and vorinostat. The kinase activity assay revealed that SRC and EGFR were highly activated in NCC-cOV1-C1 cells; the SRC inhibitor dasatinib and the EGFR inhibitor lapatinib exhibited antiproliferative effects and down-regulation of downstream signaling. The NCC-cOV1-C1 cell line will be a useful tool for basic and preclinical study of CCC, and the clinical utility of idarubicin HCl, vorinostat, dasatinib, lapatinib is worthy of further investigation.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83538150","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 : 2020-01-01DOI: 10.2198/jelectroph.64.13
Rei Noguchi, Yooksil Sin, T. Kondo
SUMMARY Protein phosphorylation is a key mechanism that regulates cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Aberrations in kinase activity and subsequent dysregulation of protein phosphorylation occur in the process of carcinogenesis and cancer progression and are considered to be therapeutic targets and biomarkers in oncology. Gel electrophoresis has versatile utility in the study of protein phosphorylation. Phosphorylated proteins can be enriched prior to gel electrophoresis, and the proteins separated by gel electrophoresis are visualized by colorimetric methods or western blotting with antibodies, which specifically detect phosphorylation. Phosphorylated proteins migrate differently from non-phosphorylated proteins in gels containing substrates with affinity for phosphorylation. All these methods can be combined in multiple ways, generating unique data from different viewpoints. The identification of separated proteins can be achieved by mass spectrometry, making it possible to integrate protein and genetic data. Peptide array allow the evaluation of kinase activity in heterogeneous samples. As protein phosphorylation and kinase activity are under the regulation of multiple mechanisms and their statuses influence the structures and functions of the other proteins, a multidisciplinary approach is required, and gel electrophoresis will play an important role in the study of protein phosphorylation.
{"title":"Gel electrophoresis for phosphorylated proteins: a brief introduction","authors":"Rei Noguchi, Yooksil Sin, T. Kondo","doi":"10.2198/jelectroph.64.13","DOIUrl":"https://doi.org/10.2198/jelectroph.64.13","url":null,"abstract":"SUMMARY Protein phosphorylation is a key mechanism that regulates cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Aberrations in kinase activity and subsequent dysregulation of protein phosphorylation occur in the process of carcinogenesis and cancer progression and are considered to be therapeutic targets and biomarkers in oncology. Gel electrophoresis has versatile utility in the study of protein phosphorylation. Phosphorylated proteins can be enriched prior to gel electrophoresis, and the proteins separated by gel electrophoresis are visualized by colorimetric methods or western blotting with antibodies, which specifically detect phosphorylation. Phosphorylated proteins migrate differently from non-phosphorylated proteins in gels containing substrates with affinity for phosphorylation. All these methods can be combined in multiple ways, generating unique data from different viewpoints. The identification of separated proteins can be achieved by mass spectrometry, making it possible to integrate protein and genetic data. Peptide array allow the evaluation of kinase activity in heterogeneous samples. As protein phosphorylation and kinase activity are under the regulation of multiple mechanisms and their statuses influence the structures and functions of the other proteins, a multidisciplinary approach is required, and gel electrophoresis will play an important role in the study of protein phosphorylation.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86985357","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}
Emiko Kinoshita-Kikuta, K. Akayama, E. Kinoshita, T. Koike
We describe a method for detecting phosphoproteins by dot-blot staining with a Phos-tag Aqua fluorescent dye. By using the method, three types of in vitro protein kinase assays for visualizing Tyr-, His-, and Asp-phosphoproteins, respectively, were performed. The staining procedure, which requires less than 2.5 h to complete, is conducted under conditions of neutral pH that are particularly advantageous for detecting labile Hisand Asp-phosphoproteins. This method promises rapid and easy detection of phosphoproteins, and should be useful in high-throughput profiling of in vitro kinase activities or in vitro kinase inhibition.
{"title":"A dot-blot-staining method for detecting phosphoproteins with a Phos-tag Aqua fluorescent dye","authors":"Emiko Kinoshita-Kikuta, K. Akayama, E. Kinoshita, T. Koike","doi":"10.2198/jelectroph.64.7","DOIUrl":"https://doi.org/10.2198/jelectroph.64.7","url":null,"abstract":"We describe a method for detecting phosphoproteins by dot-blot staining with a Phos-tag Aqua fluorescent dye. By using the method, three types of in vitro protein kinase assays for visualizing Tyr-, His-, and Asp-phosphoproteins, respectively, were performed. The staining procedure, which requires less than 2.5 h to complete, is conducted under conditions of neutral pH that are particularly advantageous for detecting labile Hisand Asp-phosphoproteins. This method promises rapid and easy detection of phosphoproteins, and should be useful in high-throughput profiling of in vitro kinase activities or in vitro kinase inhibition.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"111 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74345750","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}
When cytosolic proteins from mouse liver were separated by a combined method of native isoelectric focusing (IEF) and blue native electrophoresis, spots at pI 6.7/180,000 Da, pI 6.5/100,000 Da and pI 6.4/70,000 Da were detected by Coomassie Brilliant Blue. After separation and detection, the native enzymes were extracted by native electrophoresis and immobilized on the reverse-phase chromatography media ZipTip to produce an enzyme reactor. The hydrolysis activity of 4-methylumebelliferyl acetate by the spots at pI 6.7/180,000 Da and pI 6.4/70,000 Da was 3.0 and 2.4 times, respectively, greater than that with no enzyme. The method can be applied to systematically produce biological reactors after separation and detection of enzymes by the combined method of native IEF and blue native electrophoresis.
{"title":"Production of dye-binding esterase reactor after separation and detection using combined native isoelectric focusing and blue native electrophoresis","authors":"Y. Shimazaki, Hikari Nakamaru","doi":"10.2198/jelectroph.64.1","DOIUrl":"https://doi.org/10.2198/jelectroph.64.1","url":null,"abstract":"When cytosolic proteins from mouse liver were separated by a combined method of native isoelectric focusing (IEF) and blue native electrophoresis, spots at pI 6.7/180,000 Da, pI 6.5/100,000 Da and pI 6.4/70,000 Da were detected by Coomassie Brilliant Blue. After separation and detection, the native enzymes were extracted by native electrophoresis and immobilized on the reverse-phase chromatography media ZipTip to produce an enzyme reactor. The hydrolysis activity of 4-methylumebelliferyl acetate by the spots at pI 6.7/180,000 Da and pI 6.4/70,000 Da was 3.0 and 2.4 times, respectively, greater than that with no enzyme. The method can be applied to systematically produce biological reactors after separation and detection of enzymes by the combined method of native IEF and blue native electrophoresis.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"28 1","pages":"1-6"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83788762","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}
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