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DC 欄位 | 值 | 語言 |
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dc.contributor.author | Ting-Cheng Su | en |
dc.contributor.author | 蘇定正 | zh_TW |
dc.date.accessioned | 2021-07-01T08:11:59Z | - |
dc.date.available | 2021-07-01T08:11:59Z | - |
dc.date.issued | 2000 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75146 | - |
dc.description.abstract | HDAC1是屬於histone去乙醯脢(histone deacetylases)之一成員。而histone去乙醯脢在近年來則是被認為與基因表現的抑制有關。而在本篇論文中,選殖了HDAC1並且純化了重組蛋白以製備抗HDAC1多源性抗體。此外,藉由 pcDNA載體我們成功的在293T細胞中將有帶有FLAG epitope的HDAC1(FLAG–HDAC1)表現出來,而利用免疫螢光染色法觀察到受FLAG–HDAC1轉染後的293T細胞,FLAG-HDAC1於細胞中的位置與內生性之HDAC1是相同的皆是位於細胞核內。而在FLAG–HDAC1之去乙醯化測試中,FLAG-HDAC1的確可將[14C]乙醯基從histone上去除。 | zh_TW |
dc.description.abstract | HDAC1, a member of histone deacetylases, has been shown to correlate with the repression of the gene expression. In this work, I cloned the HDAC1, purified the recombinant protein and prepared the anti- HDAC 1 antibody. FLAG-tagged HDAC1 was expressed in 293T cells following transfection of HDAC1/pcDNA-tag plasmid. Subcellular localization of the FLAG-tagged HDAC1 was shown to be the same as endogenous HDAC1 (i.e., in the nucleus) by immunofluorescence staining. Furthermore, the FLAG tagged HDAC1 retains the deacetylase activity as demonstrated by the deacetylation of [14C] acetylated histones. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:11:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2000 | en |
dc.description.tableofcontents | 目錄………………………………………………………………………………………………Ⅰ 英文摘要…………………………………………………………………………………………..Ⅲ 中文摘要…………………………………………………………………………………………Ⅳ 第一章,緒論(Introduction)..................................................................................................................................1 第二章,材料與方法(Materials and Methods)......................................................................................................5 一,RNA 萃取及單股cDNA 制備(RNA Exraction and Single strand cDNA Preparation)................................5 二,聚合?連鎖反應(Polymerase Chain Reaction)...........................................................................................6 三,cDNA 選殖(cDNA Cloning)........................................................................................................................7 四,鹼性溶菌法之質體抽取(Plasmid Preparation)............................................................................................7 五,勝任細胞製備(Competent Cell Preparation)................................................................................................9 六,細胞轉型(Transformation)..........................................................................................................................10 七,SDS 聚丙烯醯胺凝膠電泳(SDS-PAGE).....................................................................................................10 八,GST 融合 HDAC1 重組蛋白質之表現(Expression of Recombinant GST-Fusion HDAC1 Protein)...........12 九,HDAC-l 重組蛋白之製備純化(Preparation and Purification of Recombinant HDAC-1)..........................12 十,SDS 製備型凝膠製作(Preparation of SDS-preparative Gel).........................................................................13 十一,抗 HDAC-1 多源抗體製備(Preparation of Anti-HDAC1 Polyclonal Antibody).......................................13 十二,西方點墨法(Western Blot)........................................................................................................................14 十三,細胞培養(Cell Culture)...............................................................................................................................15 十四,全細胞萃取物製備(Preparation of Whbole Cell Extract)............................................................................15 十五,細胞核萃取物製備(Preparation of Nuclear Extract)..................................................................................16 十六,間接免疫螢光染色法(Indirect Immunofluorescence).................................................................................16 十七,磷酸鈣轉染法(Calcium Phosphate Transfection)........................................................................................17 十八,銀染法(Silver Stain)....................................................................................................................................18 十九,FLAG-Tagged HDAC1之純化(Purification of Flag-Tagged HDAC1).........................................................19 二十,免疫沉澱法(Immunoprecipitation).............................................................................................................20 二一,Histone之乙醯化(Acetylation of Histone).....................................................................................................20 二二,Histone之去乙醯化(Deacetylation of Histone).............................................................................................20 第三章,結果(Results).............................................................................................................................................22 一,HDAC1 之 cDNA 選殖....................................................................................................................................22 二,HDAC1 之蛋白質表現純化與多源抗體製備................................................................................................22 三,HDAC1 於 293T 細胞之表現.........................................................................................................................24 四,FLAG-HDAC1 於 293T 細胞內分佈................................................................................................................24 五,HDAC1 之 Deacetylation 活性測試..................................................................................................................25 第四章,討論(Discussion)...........................................................................................................................................26 一,HDAC1 之蛋白質表現純化與多源抗體製備...............................................................................................26 二,FLAG-HDAC1 於 293T 細胞中之表現與位置分佈........................................................................................27 三,HDAC1 之 Deacetylation 活性測試...............................................................................................................28 四,未來之研究方向....................................................................................................................................29 參考資料......................................................................................................................................................................30 圖一:HDAC1 之選殖與其 GST 融合蛋白之表現..................................................................................................34 圖二:E.Coli中大量表現的 HDAC1之 GST 融合蛋白為不溶性蛋白.....................................................................35 圖三:TNE 及 2 % TritonX-100 能大量清洗位於 Cell Pellet 中其他蛋白質.........................................................36 圖四:GST-HDAC1 之純化....................................................................................................................................37 圖五:抗 HDAC1之多源抗之西方點墨法測試.........................................................................................................38 圖六:抗 HDAC1之多源抗之免疫沉澱法測試.......................................................................................................39 圖七:HDAC1於 Mammalian 細胞中之表現.............................................................................................................40 圖八;內生性與 FLAG–HDAC1於 293T 細胞分佈位置之比較............................................................................41 圖九:FLAG-HDAC1之 Histone Deacetylation 之活性測試........................................................................................42 | |
dc.language.iso | zh-TW | |
dc.title | Histone之去乙醯脢1(HDAC1):免疫化學與生物化學上之鑑定 | zh_TW |
dc.title | Histone Deacetylase 1(HDAC1): Immunochemical and Biochemical Characterizations | en |
dc.date.schoolyear | 88-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 50 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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