Development of an assay platform for screening Src kinase inhibitors using dot-blot assay
碩士 === 國立清華大學 === 分子與細胞生物研究所 === 104 === Src tyrosine kinase, known as a proto-oncogene, participates in many signal pathways to regulate various cellular functions. Abnormal activation of Src is also involved in cancer progression. Autophosphorylation of Src plays a crucial role in its activation....
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ndltd-TW-104NTHU50610752017-08-12T04:35:42Z http://ndltd.ncl.edu.tw/handle/90642775013174705676 Development of an assay platform for screening Src kinase inhibitors using dot-blot assay 建立以墨點法篩選Src酪胺酸激酶抑制劑的平台 Huang, Bo Yi 黃渤溢 碩士 國立清華大學 分子與細胞生物研究所 104 Src tyrosine kinase, known as a proto-oncogene, participates in many signal pathways to regulate various cellular functions. Abnormal activation of Src is also involved in cancer progression. Autophosphorylation of Src plays a crucial role in its activation. Herein, I have developed an assay platform to screen Src tyrosine kinase inhibitors by analyzing the autophosphorylation of Src. The autophosphorylation reaction was performed in a 96-well format thermocycler and the autophosphorylation of Src was analyzed by dot-blot assay. In this assay platform, the Z`-factor for each plate was greater than 0.49 and the acceptable tolerance of dimethyl sulfoxide was up to 1%. No drift or edge effects was observed. Known kinase inhibitors were used to validate this assay platform. All known Src tyrosine kinase inhibitors exhibited the inhibition of Src kinase activity in the confirmatory assay. The IC50 of Dasatinib and Saracatinib, two potent Src tyrosine kinase inhibitors, measured by this assay platform are 7.7 and 24.4 nM, respectively. Thus, this assay platform can be used to screen Src tyrosine kinase inhibitors for discovery of anti-cancer drug. Fu, Hua Wen 傅化文 2016 學位論文 ; thesis 54 en_US |
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碩士 === 國立清華大學 === 分子與細胞生物研究所 === 104 === Src tyrosine kinase, known as a proto-oncogene, participates in many signal pathways to regulate various cellular functions. Abnormal activation of Src is also involved in cancer progression. Autophosphorylation of Src plays a crucial role in its activation. Herein, I have developed an assay platform to screen Src tyrosine kinase inhibitors by analyzing the autophosphorylation of Src. The autophosphorylation reaction was performed in a 96-well format thermocycler and the autophosphorylation of Src was analyzed by dot-blot assay. In this assay platform, the Z`-factor for each plate was greater than 0.49 and the acceptable tolerance of dimethyl sulfoxide was up to 1%. No drift or edge effects was observed. Known kinase inhibitors were used to validate this assay platform. All known Src tyrosine kinase inhibitors exhibited the inhibition of Src kinase activity in the confirmatory assay. The IC50 of Dasatinib and Saracatinib, two potent Src tyrosine kinase inhibitors, measured by this assay platform are 7.7 and 24.4 nM, respectively. Thus, this assay platform can be used to screen Src tyrosine kinase inhibitors for discovery of anti-cancer drug.
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author2 |
Fu, Hua Wen |
author_facet |
Fu, Hua Wen Huang, Bo Yi 黃渤溢 |
author |
Huang, Bo Yi 黃渤溢 |
spellingShingle |
Huang, Bo Yi 黃渤溢 Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
author_sort |
Huang, Bo Yi |
title |
Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
title_short |
Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
title_full |
Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
title_fullStr |
Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
title_full_unstemmed |
Development of an assay platform for screening Src kinase inhibitors using dot-blot assay |
title_sort |
development of an assay platform for screening src kinase inhibitors using dot-blot assay |
publishDate |
2016 |
url |
http://ndltd.ncl.edu.tw/handle/90642775013174705676 |
work_keys_str_mv |
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