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研究生:古美雲
研究生(外文):Mei-Yun Ku
論文名稱:利用二維電泳法分析鼠腦之後突觸質密區(PSD)與其主要蛋白α-CaMKⅡ
論文名稱(外文):Analysis of postsynaptic density and α-CaMKⅡ in rat brain by two-dimensional gel electrophoresis
指導教授:張兗君
指導教授(外文):Yen-Chung Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:81
中文關鍵詞:二維電泳後突觸質密區磷酸化酵素麩胺酸受器西方點漬法銀染色法
外文關鍵詞:two-dimensional gel electrophoresispostsynaptic densityα-CaMKⅡglutamate receptorWestern BlotSliver stain
相關次數:
  • 被引用被引用:1
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  • 下載下載:21
  • 收藏至我的研究室書目清單書目收藏:0
Postsynaptic density (PSD) 是一個細緻的圓盤狀結構,它位於後突觸膜的下方,與調控後突觸的訊息傳遞有關,並且經由調控突觸區域的活性,PSD被相信與學習和記憶有所關聯。之前有許多研究,利用不同的方法來研究PSD的組成,發現其中含有多種蛋白,包括glutamate receptors、 scaffold proteins 和 signaling proteins等等,近年來利用高效率及高解析度的質譜儀系統,現已分析出近500種PSD的組成蛋白。我們實驗室利用不同pH值範圍與不同濃度的二維電泳膠片,分做四個區域來研究PSD組成蛋白在二維膠片上的分布,發現到有超過600個具再現性protein spots的表現,相信可能仍有許多未知的PSD組成蛋白尚未被發現。而我們在二維電泳方面的研究,亦大為改善PSD組成蛋白在鹼性pI值與低分子量區域的分析效果。另外,借助MALDI-TOF的分析,我們做了八個major protein spots的鑑定,發現它們表現在2-D gel的pI/MW值與MW的實際值和pI的理論計算值都很接近。α-CaMKⅡ是PSD中的主要蛋白之一,過去的研究沒有明確的找出它在2-D gel上的位置,我們原本想利用二維電泳配合Western Blot做α-CaMKⅡ的定位,卻發現α-CaMKⅡ在不同pH值的溶液中,表現在膠片上的量不同。於是我們做了溶解度測試的實驗,發現pH6-11 IPG buffer是促成α-CaMKⅡ有效溶解的主因。未來我們可以利用pH6-11 IPG buffer配合改良的二維電泳條件,以期正確定位出α-CaMKⅡ在2-D gel的位置,這將有助於了解α-CaMKⅡ不同的磷酸化狀態,並且推測α-CaMKⅡ是如何調控與PSD之間的關係。
摘要
壹、 前言……………………………………………………………….1
貳、 材料與方法……………………………………………….……..10
(一) 藥品與材料 ……………………………..…………….…….10
(二) PSD的純化…………………………………….………….…10
(三) 蛋白質濃度測定……………………………………….……..14
(四) 二維電泳法(two-dimensional gel electrophoresis)…………..14
(五) 銀染色法(Silver stain)……………………………….…….…18
(六) Coomassie Blue染色法…………………………..…………..19
(七) ProtoGold染色法……………………………………………..20
(八) 膠體內蛋白質分解(In-gel digestion for silver stain)……...…20
(九) 去染色(destain) 和西方點漬法(Western Blot)……………...21
參、 結果………………………………………………………………23
(一) 在pH4-7,8.5% 2-D gel的部分…………………………...23
(二) 在pH4-7,12% 2-D gel的部分……………………………23
(三) 在pH6-11,8.5% 2-D gel的部分……………………….…24
(四) 在pH6-11,12% 2-D gel的部分…………………………..25
(五) 四個區域2-D gel上protein spots的統計………………..….26
(六) protein spots的鑑定…………………………………………..26
(七) 利用western blot找出α-CaMKⅡ在2-D gel上的位置…….27
(八) 在pH4-7 rehydration solution和pH6-11 lysis solution中α-CaMKⅡ的溶解度不同……………………………….……28
(九) pH 6-11 IPG buffer是提高α-CaMKⅡ溶解度的主因………29
肆、 討論…………………………………………………………..…..31
(一) PSD組成蛋白的研究…………………………………….…...31
(二) α-CaMKⅡ的溶解度對於二維電泳分析的限制………..……33
伍、參考文獻………………………………………………………..…35
圖表集……………………………………………………………..……45
Appendix Ⅰ…………………………………………………………….77
Appendix Ⅱ…………………………………………………………….78
Appendix Ⅲ………………………………………………………….…79
Appendix Ⅳ…………………………………………………………….80
Appendix Ⅴ……………………………………………...……………..81
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