Development of an assay platform for screening Src kinase inhibitors using dot-blot assay

• Main Authors: Huang, Bo Yi, 黃渤溢
• Other Authors: Fu, Hua Wen
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/90642775013174705676

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.