Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin
The electrophoretic method of Schägger and von Jagow ( Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated...
Gespeichert in:
| Veröffentlicht in: | Analytical biochemistry 1995-10, Vol.231 (1), p.218-224 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 224 |
|---|---|
| container_issue | 1 |
| container_start_page | 218 |
| container_title | Analytical biochemistry |
| container_volume | 231 |
| creator | Klement, P. Nijtmans, L.G.J. Vandenbogert, C. Houstek, J. |
| description | The electrophoretic method of Schägger and von Jagow (
Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10
6 fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders. |
| doi_str_mv | 10.1006/abio.1995.1523 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_77770192</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S0003269785715230</els_id><sourcerecordid>77770192</sourcerecordid><originalsourceid>FETCH-LOGICAL-c339t-545216d62239a288b7c64e35a72221e8b1eed728e00b1b114ccbee43e6e771183</originalsourceid><addsrcrecordid>eNp1kT1vFDEQhq0IFI6Qlg7JFd0e_tjP8jiSXKRAKEht2d65nJHXXmxvyP4rfmK83ImOaUajeecZe16E3lOypoTUn6Qyfk27rlrTivEztKKkqwvCSfcKrQghvGB117xBb2P8SQilZVWfo_O2blpOyhX6s3HSztFE7Pf4_tn0MpknwN8PPo4HH2aba-_w1g-jhWeI2ORismkK0OPdNEiHr40KXlkZU8TS9XgzOOP1nLJYzfiznaD49pdaXFnQKfgFDMvKh2jcI_5qkh-P47fR54WZ_NukA04HwDuw4_K0L-Yxy5xx79DrvbQRLk_5Aj1cX_3Y7oq7-5vb7eau0Jx3qajKitG6rxnjnWRtqxpdl8Ar2TDGKLSKAvQNa4EQRVU-i9YKoORQQ9NQ2vIL9PHIHYP_NUFMYjBRg7XSgZ-iaHIQ2rEsXB-FOvgYA-zFGMwgwywoEYtFYrFILBaJxaI88OFEntQA_T_5yZPcb499yN97MhBE1Aacht6EfD_Re_M_9AtQo6Pt</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>77770192</pqid></control><display><type>article</type><title>Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin</title><source>MEDLINE</source><source>ScienceDirect Journals (5 years ago - present)</source><creator>Klement, P. ; Nijtmans, L.G.J. ; Vandenbogert, C. ; Houstek, J.</creator><creatorcontrib>Klement, P. ; Nijtmans, L.G.J. ; Vandenbogert, C. ; Houstek, J.</creatorcontrib><description>The electrophoretic method of Schägger and von Jagow (
Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10
6 fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.1995.1523</identifier><identifier>PMID: 8678304</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amniotic Fluid - metabolism ; Cells, Cultured ; Digitonin ; Electron Transport ; Electrophoresis, Polyacrylamide Gel - methods ; Enzymes - analysis ; Female ; Fibroblasts - metabolism ; Humans ; Mitochondria - metabolism ; Oxidative Phosphorylation ; Pregnancy ; Skin - metabolism</subject><ispartof>Analytical biochemistry, 1995-10, Vol.231 (1), p.218-224</ispartof><rights>1995 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c339t-545216d62239a288b7c64e35a72221e8b1eed728e00b1b114ccbee43e6e771183</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/abio.1995.1523$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8678304$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Klement, P.</creatorcontrib><creatorcontrib>Nijtmans, L.G.J.</creatorcontrib><creatorcontrib>Vandenbogert, C.</creatorcontrib><creatorcontrib>Houstek, J.</creatorcontrib><title>Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>The electrophoretic method of Schägger and von Jagow (
Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10
6 fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.</description><subject>Amniotic Fluid - metabolism</subject><subject>Cells, Cultured</subject><subject>Digitonin</subject><subject>Electron Transport</subject><subject>Electrophoresis, Polyacrylamide Gel - methods</subject><subject>Enzymes - analysis</subject><subject>Female</subject><subject>Fibroblasts - metabolism</subject><subject>Humans</subject><subject>Mitochondria - metabolism</subject><subject>Oxidative Phosphorylation</subject><subject>Pregnancy</subject><subject>Skin - metabolism</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kT1vFDEQhq0IFI6Qlg7JFd0e_tjP8jiSXKRAKEht2d65nJHXXmxvyP4rfmK83ImOaUajeecZe16E3lOypoTUn6Qyfk27rlrTivEztKKkqwvCSfcKrQghvGB117xBb2P8SQilZVWfo_O2blpOyhX6s3HSztFE7Pf4_tn0MpknwN8PPo4HH2aba-_w1g-jhWeI2ORismkK0OPdNEiHr40KXlkZU8TS9XgzOOP1nLJYzfiznaD49pdaXFnQKfgFDMvKh2jcI_5qkh-P47fR54WZ_NukA04HwDuw4_K0L-Yxy5xx79DrvbQRLk_5Aj1cX_3Y7oq7-5vb7eau0Jx3qajKitG6rxnjnWRtqxpdl8Ar2TDGKLSKAvQNa4EQRVU-i9YKoORQQ9NQ2vIL9PHIHYP_NUFMYjBRg7XSgZ-iaHIQ2rEsXB-FOvgYA-zFGMwgwywoEYtFYrFILBaJxaI88OFEntQA_T_5yZPcb499yN97MhBE1Aacht6EfD_Re_M_9AtQo6Pt</recordid><startdate>19951010</startdate><enddate>19951010</enddate><creator>Klement, P.</creator><creator>Nijtmans, L.G.J.</creator><creator>Vandenbogert, C.</creator><creator>Houstek, J.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19951010</creationdate><title>Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin</title><author>Klement, P. ; Nijtmans, L.G.J. ; Vandenbogert, C. ; Houstek, J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c339t-545216d62239a288b7c64e35a72221e8b1eed728e00b1b114ccbee43e6e771183</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>Amniotic Fluid - metabolism</topic><topic>Cells, Cultured</topic><topic>Digitonin</topic><topic>Electron Transport</topic><topic>Electrophoresis, Polyacrylamide Gel - methods</topic><topic>Enzymes - analysis</topic><topic>Female</topic><topic>Fibroblasts - metabolism</topic><topic>Humans</topic><topic>Mitochondria - metabolism</topic><topic>Oxidative Phosphorylation</topic><topic>Pregnancy</topic><topic>Skin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Klement, P.</creatorcontrib><creatorcontrib>Nijtmans, L.G.J.</creatorcontrib><creatorcontrib>Vandenbogert, C.</creatorcontrib><creatorcontrib>Houstek, J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Klement, P.</au><au>Nijtmans, L.G.J.</au><au>Vandenbogert, C.</au><au>Houstek, J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1995-10-10</date><risdate>1995</risdate><volume>231</volume><issue>1</issue><spage>218</spage><epage>224</epage><pages>218-224</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>The electrophoretic method of Schägger and von Jagow (
Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10
6 fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>8678304</pmid><doi>10.1006/abio.1995.1523</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 1995-10, Vol.231 (1), p.218-224 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_77770192 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Amniotic Fluid - metabolism Cells, Cultured Digitonin Electron Transport Electrophoresis, Polyacrylamide Gel - methods Enzymes - analysis Female Fibroblasts - metabolism Humans Mitochondria - metabolism Oxidative Phosphorylation Pregnancy Skin - metabolism |
| title | Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-13T02%3A05%3A04IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Analysis%20of%20Oxidative%20Phosphorylation%20Complexes%20in%20Cultured%20Human%20Fibroblasts%20and%20Amniocytes%20by%20Blue-Native-Electrophoresis%20Using%20Mitoplasts%20Isolated%20with%20the%20Help%20of%20Digitonin&rft.jtitle=Analytical%20biochemistry&rft.au=Klement,%20P.&rft.date=1995-10-10&rft.volume=231&rft.issue=1&rft.spage=218&rft.epage=224&rft.pages=218-224&rft.issn=0003-2697&rft.eissn=1096-0309&rft_id=info:doi/10.1006/abio.1995.1523&rft_dat=%3Cproquest_cross%3E77770192%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=77770192&rft_id=info:pmid/8678304&rft_els_id=S0003269785715230&rfr_iscdi=true |