Multi-dimension polyacrylamide gel electrophoresis for detection of metal ions bound to metalloproteins
| Project/Area Number |
23750077
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Analytical chemistry
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| Research Institution | Saitama University |
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Principal Investigator |
SAITO Shingo 埼玉大学, 大学院・理工学研究科, 准教授 (60343018)
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2012: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2011: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
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| Keywords | 分離分析 / 金属タンパク質 / ゲル電気泳動法 / 蛍光プローブ / メタロタンパク質 / 金属蛍光プローブ / ポリアクリルアミドゲル電気泳動 / スペシエーション |
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| Research Abstract |
A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two gel electrophoresis, to obtain distribution of protein-bound metal ions in biological samples. The former is blue-native (BN) PAGE to separate proteins without dissociation during migration. The other is a PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using fluorescence metal probe. The probes have a chelating moiety (multidentate polyaminocarboxylate), a fluorophore (fluorescein) and a spacer (aminobenzyl group) to form emissive and kinetic inert complexes with metal ions. Some metal ions, Mg^<2+>, Cu^<2+>, Fe^<2+>/^<3+>, Ni^<2+>, Co^<2+>, Mn^<2+>, Cd^<2+> and Hg^<2+>, were successfully detected by this new PAGE. After separation of the proteins by BN-PAGE, the metal ions in the gel fractions were eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxidedismutase, and iron ions bound to transferrin and hemopexin were quantitatively determined.
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Report
(3 results)
Research Products
(29 results)
